KR101993254B1 - Angiogenesis using placental stem cells - Google Patents

Abstract

Methods of treating placental cells, such as placental stem cells and placental pluripotent cells (PDACs) described herein, and individuals having a disease or disorder in the circulatory system using the placental cell population are provided herein. The present invention also provides an angiogenesis method using the cell or a population of cells comprising the cell.

Description

[0001] ANGIOGENESIS USING PLACENTAL STEM CELLS [0002]

1. Field

Tissue culture Plastics-adherent placental cells, such as placental stem cells (referred to herein as PDACs), are used to promote angiogenesis and to treat diseases or disorders of the circulatory system, such as those associated with or resulting from inappropriate angiogenesis or blood flow Methods for treating a disease or disorder that is treatable by enhancing angiogenesis or a resulting disease or disorder are provided herein.

2. Background

The placenta is a particularly attractive source of stem cells. Since the mammalian placenta is abundant and normally disposed of as medical waste, these represent the only source of medically useful stem cells. Such isolated placental stem cells, a population of placental stem cells, and methods of using the same to treat diseases or disorders that promote angiogenesis and treat diseases or disorders of the circulatory system, such as by treatment of angiogenesis, are provided herein do.

3. Overview

In one aspect, a therapeutically effective amount of a tissue culture plastic-adherent placental cell, such as a placental stem cell (also referred to herein as PDAC) (a placenta-derived adherent cell, for example, A placenta-derived adherent cell described in Section 5.2) in a sufficient amount and for a sufficient time for a detectable improvement of one or more symptoms of the disease or disorder. Are provided herein. However, the PDAC described herein is a pending U.S. Patent Application No. 12 / 622,352 entitled " Amnion Derived Angiogenic Cells ", filed on November 19, 2009, 0.0 > adherent < / RTI > adherent cells described in US Pat. In certain embodiments, the method comprises administering to the subject a PDAC, a chest cardiac output (CO), cardiac index (CI), pulmonary wedge pressure (PAWP), cardiac index (CI),% % FS), extrusion coefficient (EF), left ventricular ejection fraction (LVEF); (LVEDD), left ventricular end-diastolic diameter (LVESD), contractility (dP / dt), decrease in atrial or ventricular function, increase in pumping efficiency, decrease in pumping efficiency loss rate, In a sufficient amount and for a sufficient time for a detectable improvement of one or more symptoms of cardiac function which is a decrease in complications associated with cardiomyopathy.

In another embodiment, the disease or disorder of the circulatory system is myocardial infarction. In another embodiment, the disease or disorder is cardiomyopathy. In another embodiment, the disease or disorder is selected from the group consisting of aneurysm, angina pectoris, aortic stenosis, aortitis, arrhythmia, arteriosclerosis, arteritis, asymmetric heavy wall hyperplasia (ASH), atherosclerosis, atrial fibrillation and coarctation, bacterial endocarditis, Atherosclerosis, congestive heart failure, coronary artery disease, Eisenmenger syndrome, embolism, endocarditis, cardiomyopathy, cardiomyopathy, cardiomyopathy, cardiac arrhythmia, congestive heart failure, congestive heart failure, Kawasaki disease (mucosal skin lymphadenopathy, mucosal skin lymphadenopathy, infantile multiple arteritis), metabolic syndrome, microvessels, acute myelogenous leukemia, skin inflammation, fibrillation, fibrous dysplasia, cardiac arrest, cardiac noise, hypertension, hypotension, idiopathic infant artery calcification Angina pectoris, myocarditis, paroxysmal atrial tachycardia (PAT), nodular arteriosclerosis (multiple arteritis, nodular polyarteritis), pericarditis, peripheral vascular disease Atherosclerosis, renal artery stenosis, renovascular hypertension, rheumatic heart disease, diabetic angiopathy, septal defect, asymptomatic ischemia, syndrome X, tachyarrhythmia, tachyarrhythmia, Parkinson-White syndrome, intimal hyperplasia, acute rheumatic fever, acute rheumatic fever, acute rheumatic fever, acute rheumatic fever, acute rheumatic fever, acute rheumatic fever, acute rheumatic fever, rheumatoid arthritis, Acute rheumatic myocarditis, chronic rheumatic heart disease, mitral valve disease, mitral stenosis, rheumatic mitral regurgitation, aortic valve disease, diseases of other endocardial structures, ischemic heart disease (acute and subacute), rheumatoid arthritic endocarditis, acute rheumatic endocarditis, , Chest tightness, acute pulmonary heart disease, pulmonary embolism, chronic pulmonary heart disease, posterior scoliosis heart disease, myocarditis, endocarditis, Muscle fibrosis, endocardial elastic fibers is a syndrome, atrioventricular block, heart rhythm disorders, myocardial degeneration, cerebrovascular disease, arteries, arterioles and capillaries in diseases or disorders of the veins and lymphatic vessels.

In another specific embodiment, the disease or disorder is occlusion and stenosis of the brainstem artery, or occlusion of the cerebral artery. In one aspect, there is provided a method of treating a subject suffering from a symptom or neurological deficit resulting from the destruction of blood flow in or around the brain of an individual, for example, the brain or central nervous system (CNS) Methods of treating such individuals, including administering an effective amount of isolated placental stem cells (e. G., PDAC) are provided herein. In certain embodiments, destruction of blood flow causes anoxic or hypoxic damage to the brain or CNS of an individual.

In another specific embodiment, the disease or disorder is occlusion and stenosis of the peripheral artery. In one aspect, administration of a therapeutically effective amount of an isolated PDAC to a subject suffering from a destruction of blood flow in or around the limb, e.g., a subject suffering from a symptom or vascular defect resulting from the destruction of blood flow in or around the peripheral vasculature of an individual A method of treating said subject is provided herein. In certain embodiments, destruction of blood flow causes oxygen free or hypoxic damage to the extremities and / or limbs of an individual.

In another specific embodiment of the method of treatment, said cells are administered to said subject by injection. In a more specific embodiment, the injection is an injection into the ischemic area of the subject's heart. In another specific embodiment of the method of treatment, said cells are administered to said subject by intravenous infusion. In another specific embodiment of the method of treatment, said cell, or a population of such cells, or a population of cells comprising said cells, is administered by inserting a matrix or scaffold comprising placenta-derived adherent cells as described above into said subject Is administered to the subject.

In certain embodiments, the placental cells are administered intravenously to the subject. In another embodiment, the method of treatment comprises administering at least about 1 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ , 5 x 10 ⁸ , 1 x 10 ⁹ , 5 x 10 ⁹ , or 1 x 10 ¹⁰ placental cells ≪ / RTI > In another specific embodiment, the placental cells described herein are administered at a dose of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, , 44, 45, 46, 47, 48, 49, or 50 groups. In another specific embodiment, any of the placental cells described herein is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, or 20 or more times. In another embodiment of any of the embodiments herein, the placental cells are cryopreserved and thawed prior to administration.

In another embodiment, the placental cells are cryopreserved and thawed prior to contacting.

In certain embodiments of any of the above methods, the placental cells are adherent to tissue culture plastic, when detected by a flow cytometry CD34 ^-, the CD10 ^+, CD105 ⁺ and CD200 ^+. In certain embodiments, placental cells have the ability to differentiate into osteogenic or cartilage forming cells. In another embodiment, the placental cell is adherent to tissue culture plastic; CD34 ^- , CD10 ⁺ , CD105 ⁺ and CD200 ⁺ upon detection by flow cytometry; Have the ability to differentiate into osteogenic or cartilage forming cells with one or more characteristics, for example bone cells or chondrocyte cells. In another embodiment, the placental cell further comprises at least one characteristic of a neuron or neuron cell, for example a characteristic of a neuron; One or more characteristics of glial cells, for example, the characteristics of glial cells or astrocytes; One or more characteristics of adipocyte cells, such as the characteristics of adipocytes; One or more characteristics of pancreatic cells; And / or cells having one or more characteristics of cardiac cells.

In another embodiment, the placental cells are selected from the group consisting of CD34 ^- , CD10 ⁺ , CD105 ⁺ and CD200 ⁺ , and CD38 ^- , CD45 ^- , CD80 ^- , CD86 ^- , CD133 ^- , HLA-DR, DP, DQ -, SSEA3 -, SSEA4 -, CD29 +, CD44 +, CD73 +, CD90 +, CD105 +, HLA-A, B, C +, PDL1 +, ABC-p + and / or OCT-4 ⁺ . ^& Lt; / RTI > In another embodiment, the placental cells are CD34 upon detection by flow cytometry ^-, CD45 ^-, the CD10 ^+, CD90 ^+, CD105 ⁺ and CD200 ^+. In another embodiment, the placental cells are CD34 upon detection by flow cytometry ^-, CD45 ^-, CD10 ^+, CD80 ^-, CD86 ^-, the CD90 ^+, CD105 ⁺ and CD200 ^+. In another embodiment, the placental cells, when detected by a flow cytometry CD34 ^-, CD45 ^-, CD10 ^+, CD80 ^-, CD86 ^-, CD90 ^+, CD105 ⁺ and CD200 ^+, and further CD29 ^+, with CD38 ^-, CD44 ⁺ , CD54 ⁺ , SH3 ⁺ or SH4 ⁺ . In another embodiment, the placental cells are CD34 upon detection by flow cytometry ^-, CD38 ^-, CD45 ^-, CD10 ^+, CD29 ^+, CD44 ^+, CD54 ^+, CD73 ^+, CD80 ^-, CD86 ^-, CD90 ^+, CD105 ⁺ and CD200 ⁺ .

In another embodiment, the CD34 ^-, CD10 ^+, CD105 ⁺ and CD200 ⁺ placental cells are added to the ^{CD117 -, CD133 -, KDR -} (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ , DR ^-, or a programmed death ligand -1 (PDL1) ⁺ one or more, or a combination of any of the. , CD38 ^{- -} In yet another specific embodiment, it said placental cells are CD34 upon detection by flow cytometry, CD45 ^-, CD10 ^+, CD29 ^+, CD44 ^+, CD54 ^+, CD73 ^+, CD80 ^-, CD86 ^-, CD90 ^{+ , CD105 +, CD117 -, CD133} -, CD200 +, KDR - (VEGFR2 -), HLA-a, B, C +, HLA-DP, DQ, DR -, or programmed death ligand -1 (PDL1) ⁺ to be.

In another embodiment, any of the placental cells described herein is additionally detected by ABC-p ⁺ , or OCT-4 ⁺ (POU5F1 ⁺ ) as determined by RT-PCR, for example by flow cytometry, ABC-p is a placenta-specific ABC transporter protein (also known as breast cancer resistant protein (BCRP) and mitoxantrone resistant protein (MXR)). In another embodiment, the additional random placental cells described herein, for when g determined by flow cytometry SSEA3 ^- or SSEA4 ^- specific, wherein SSEA3 is a stage-specific embryonic antigen-3, SSEA4 the stages Embryonic antigen 4. In another embodiment, any placental cell described herein is additionally SSEA3 ^- and SSEA4 ^- .

In another embodiment of the methods described herein, any of the placental cells described herein may additionally comprise one or more of MHC-I ⁺ (e.g. HLA-A, B, C ⁺ ), MHC-II ^- -DP, DQ, DR ^- at least one ^-) or HLA-G. In another embodiment, each MHC-I ⁺ additionally any placental cells described herein (e.g., HLA-A, B, C ^+), MHC-II ^- (e.g., HLA-DP, DQ , ^{DR-a -),} and HLA-G.

In other embodiments, CD34 ^- at least one of, or SH3 ^{+ SH4 + -, CD10 +, CD105} +, CD200 + cells is more CD29 ^+, CD38 as ^{-, CD44 +, CD54 +,} CD80 -, CD86. In another embodiment, the cell is further CD44 ⁺ . In other ^{embodiments, CD34 -, CD10 +, CD105} +, CD200 + placental cells are added to the ^{CD13 +, CD29 +, CD33 +} , CD38 -, CD44 +, CD45 -, CD54 +, CD62E -, CD62L -, CD62P ^{-, SH3 + (CD73 +)} , SH4 + (CD73 +), CD80 -, CD86 -, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD117 -, CD144 / VE- cadherin ^low, CD184 / ^{CXCR4 -, CD133 -, OCT-} 4 +, SSEA3 -, SSEA4 -, ABC-p +, KDR - (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ, DR -, HLA-G ^- , or one or more of the programmed apoptosis-1 ligand (PDL1) ⁺ , or any combination thereof. In other ^{embodiments, CD34 -, CD10 +, CD105} +, CD200 + placental cells are added to the ^{CD13 +, CD29 +, CD33 +} , CD38 -, CD44 +, CD45 -, CD54 / ICAM +, CD62E -, CD62L - ^{, CD62P -, SH3 + (CD73} +), SH4 + (CD73 +), CD80 -, CD86 -, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD117 -, CD144 / VE- cadherin ^{about, CD184 / CXCR4 -, CD133 -,} OCT-4 +, SSEA3 -, SSEA4 -, ABC-p +, KDR - (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ, DR -, HLA -G < ^- & gt ^; , and the programmed death-1 ligand (PDL1) ⁺ .

In another embodiment of the methods disclosed herein, the isolated placental stem cells are CD200 ^+, and HLA-G ^-; CD73 ⁺ , CD105 ⁺ , and CD200 ⁺ ; CD200 ⁺ and OCT-4 ⁺ ; CD73 ^+, CD105 ^+, and HLA-G ^-; CD73 ⁺ and CD105 ⁺ ; Or OCT-4 ⁺ ; Or any combination thereof.

In certain embodiments of the methods disclosed herein, the isolated placental stem cells are CD10 ^+, CD29 ^+, CD34 ^-, CD38 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ^+, SH4 ^+, SSEA3 ^-, SSEA4 ^-, and OCT-4 ^+, MHC-I ⁺ or ABC-p ⁺ one or more, where ABC-p is a placenta-specific ABC transporter protein (breast cancer resistant protein (BCRP) and mitoxantrone resistance protein (MXR)). In another embodiment, the isolated placental stem cells are CD10 ^+, CD29 ^+, CD34 ^-, CD38 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ^+, SH4 ^+, SSEA3 ^-, SSEA4 ^- , And OCT-4 ⁺ . In another embodiment, the isolated placental stem cells are ^{CD10 +, CD29 +, CD34 -} , CD38 -, CD45 -, the ^{CD54 +, SH2 +, SH3 +} , SH4 ⁺ and. In another embodiment, the isolated placental stem cells are ^{CD10 +, CD29 +, CD34 -} , CD38 -, CD45 -, the ^{CD54 +, SH2 +, SH3 +} , SH4 + and OCT-4 ^+. In another embodiment, the isolated placental stem cells are CD10 ^+, CD29 ^+, CD34 ^-, CD38 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, MHC-1 ^+, SH2 ^+, an SH3 ^+, SH4 ⁺ . In another embodiment, the isolated placental stem cells are OCT-4 ⁺ and ABC-p ⁺ . In another embodiment, the isolated placental stem cells are SH2 ⁺ , SH3 ⁺ , SH4 ⁺ and OCT-4 ⁺ . In another embodiment, the isolated placental stem cells are ^{OCT-4 +, CD34 -,} SSEA3 -, and SSEA4 ^- a. In a specific embodiment, said ^{OCT-4 +, CD34 -,} SSEA3 -, and SSEA4 ^- cells were more CD10 ^+, CD29 ^+, CD34 as ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ⁺ , And SH4 ⁺ . In another embodiment, the isolated placental stem cells are OCT-4 ⁺ and CD34 ^- a, and SH3 or SH4 ^{+ +.} In another embodiment, the isolated placental stem cells are CD34 ^-, and CD10 ^+, the ^{CD29 +, CD44 +, CD54 +} , CD90 + or OCT-4 ^+. In certain embodiments, the isolated placental stem cells are CD10 ⁺ , CD34 ^- , CD105 ⁺ and CD200 ⁺ .

In another embodiment, useful for the isolated placental stem cells with the methods described herein are ^{CD10 +, CD29 -, CD44 +} , CD45 -, CD54 / ICAM -, CD62-E -, CD62-L -, CD62-P -, ^{CD80 -, CD86 -, CD103 -} , CD104 -, CD105 +, CD106 / VCAM +, CD144 / VE- cadherin ^about, CD184 / CXCR4 ^-, β2- microglobulin ^about, MHC-I ^about, MHC-II ^-, HLA -G is ^about, and / or PDL1 one or more of the ^drugs. In certain embodiments, such placental cells are at least CD29 ^- and CD54 ^- . In another embodiment, such isolated placental stem cells are at least CD44 ⁺ and CD106 ⁺ . In another embodiment, such isolated placental stem cells are at least CD29 ⁺ .

In certain embodiments of any of the above characteristics, the expression of a cell marker (e. G., A differentiated population or immunogenic marker) is determined by flow cytometry. In another particular embodiment, the expression of the cell marker is determined by RT-PCR.

In another embodiment, the placental cells, such as the CD10 ⁺ , CD34 ^- , CD105 ⁺ , CD200 ⁺ cells, useful in the methods disclosed herein are capable of detecting one or more genes from the same number of bone marrow- derived mesenchymal stem cells Wherein the at least one gene is selected from the group consisting of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and ZC3H12A, and the bone marrow-derived mesenchymal stem cells underwent the same number of subculture cultures as the number of subcultures that the isolated placental stem cells underwent. In certain embodiments, the expression of the one or more genes is determined by, for example, RT-PCR or microarray analysis using, for example, the U133-A microarray (Affymetrix). In another embodiment, the isolated placental stem cells are cultured in a medium containing 60% DMEM-LG (e.g. from Gibco) and 40% MCDB-201 (from Sigma); 2% fetal bovine serum (e.g., from Hyclone Labs.); 1x insulin-transferrin-selenium (ITS); 1x linoleic acid-bovine serum albumin (LA-BSA); 10 ^-9 M dexamethasone (e.g., from Sigma); 10 ^-4 M ascorbic acid 2-phosphate (e.g., from Sigma); Epidermal growth factor 10 ng / mL (e. G., From R & D Systems); And from 10 ng / mL of platelet-derived growth factor (PDGF-BB) (for example, from R & D Systems), for example from about 3 to about 35 population doublings, Expresses one or more genes. In another embodiment, said isolated placental stem cells are selected from the group consisting of 60% DMEM-LG (e.g. from Gibco) and 40% MCDB-201 (e.g. from Sigma); 2% fetal bovine serum (e.g., from Hyclone Labs.); 1x insulin-transferrin-selenium (ITS); 1x linoleic acid-bovine serum albumin (LA-BSA); 10 ^-9 M dexamethasone (e.g., from Sigma); 10 ^-4 M ascorbic acid 2-phosphate (Sigma); Epidermal growth factor 10 ng / mL (from, for example, R & D Systems); When cultured for about 3 to about 35 population doublings in a medium containing 10 ng / mL of platelet-derived growth factor (PDGF-BB) (for example, from NAND Systems).

In certain embodiments, the placental cells are selected from the group consisting of CD200 and ARTSl (an aminopeptidase modulator of type 1 tumor necrosis factor); ARTS-1 and LRAP (white blood cell-derived arginine aminopeptidase); IL6 (interleukin-6) and TGFB2 (transforming growth factor, beta 2); IL6 and KRT18 (keratin 18); IER3 (extreme early response 3), MEST (mesodermal-specific transcript homolog) and TGFB2; CD200 and IER3; CD200 and IL6; CD200 and KRT18; CD200 and LRAP; CD200 and MEST; CD200 and NFE2L3 (nuclear factor (red blood cell-derived 2) -like 3); Or CD200 and TGFB2 at a detectably higher level than the same number of bone marrow-derived mesenchymal stem cells (BM-MSCs), wherein said bone marrow-derived mesenchymal stem cells are cultured in a subculture of the subculture of said placental stem cells The same number of passages as in the number of passages underwent cultivation. In another embodiment, the placental cells are ARTS-1, CD200, IL6 and LRAP; ARTS-1, IL6, TGFB2, IER3, KRT18 and MEST; CD200, IER3, IL6, KRT18, LRAP, MEST, NFE2L3 and TGFB2; ARTS-1, CD200, IER3, IL6, KRT18, LRAP, MEST, NFE2L3 and TGFB2; Or IER3, MEST and TGFB2 at a detectably higher level than the same number of bone marrow-derived mesenchymal stem cell BM-MSCs, wherein said bone marrow-derived mesenchymal stem cells are cultured in a subculture The same number of passages as in the number of passages underwent cultivation.

When human cells are used, the genetic designation refers to the human sequence throughout, and representative sequences as is well known to those skilled in the art can be found in the literature or in the GenBank. Probes for the sequences may be obtained by publicly available sequences or through commercial sources, for example, through the use of a specific TAQMAN < (R) > probe or a Taxan angiogenic array (Applied Biosystems, part no. 4378710) Can be determined.

In various embodiments, the isolated placental stem cells, e. G., Placental stem cells or placental pluripotent cells, useful in the methods disclosed herein, are at least 10%, 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% %. In another specific embodiment, the placental cells in the population of cells are substantially free of cells having a maternal genotype, for example at least 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the placental cells have the fetal genotype; In another particular embodiment, the population of cells comprising placental cells is substantially free of cells having a maternal genotype, for example at least 40%, 45%, 50%, 55%, 60% Cells of 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% have the fetal genotype; In another particular embodiment, the population of cells comprising the placental cells comprises cells having a maternal genotype; For example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 80%, 85%, 90%, 95%, 98% or 99% of the cells have a maternal genotype, that is, a maternal origin.

In an embodiment of any of the embodiments of placental cells (e. G., PDAC) of the present disclosure, the placental cells are cultured when the population is cultured under conditions permitting the formation of embryo-analogs, for example when cultured under propagation conditions Promoting the formation of one or more embryo-analogs within a population of placental cells comprising said isolated placental cells.

In certain embodiments, placental cells (e.g., PDAC) useful in the methods provided herein do not express CD34 upon detection by immunological localization after 4 to 21 days exposure to 1 to 100 ng / mL VEGF . In a specific embodiment, the placental adherent cells are adherent to tissue culture plastics. In another specific embodiment, the population of cells comprises an angiogenic factor such as vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), platelet derived growth factor PDGF) or basic fibroblast growth factor (bFGF).

In yet another aspect, at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of the cells in the PDAC, cell population, e.g. PDAC population, PDAC-BB, TIMP-1, PDGF-BB, and TIMP- 2, uPAR, or galectin-1, for example, into a culture medium in which cells or cells grow. In another embodiment, In another embodiment, the PDAC is under normal oxygen conditions (e. G., About 20% or about 21% O ₂₎ and the low-oxygen conditions as compared to (e. G., About 5% O less than ₂₎ CD202b, IL-8 and / or VEGF.

In another embodiment, a population of cells comprising any of the PDACs or PDACs described herein can cause the formation of shoots or tubular-like structures in a population of endothelial cells contacting the placenta-derived adherent cells. In a specific embodiment, the placenta-derived angiogenic cells are co-cultured with human endothelial cells and cultured for at least 4 days in or on a substrate such as placenta collagen or Matrigel < RTI ID = 0.0 > And / or an angiogenic factor such as vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), platelet derived growth factor (PDGF) or basic fibroblast growth factor (bFGF) Like structure or support endothelial cell germination.

In certain embodiments of any placental cells disclosed herein, the cell is a human cell.

In certain embodiments, any of the placental cells described herein, such as placental stem cells or placental pluripotent cells, are autologous to the recipient. In certain other embodiments, any of the placental cells described herein, such as placental stem cells or placental pluripotent cells, are of heterogeneous origin for the recipient.

In certain embodiments, placental cells are cryopreserved prior to the administration. In another embodiment, the isolated placental cells are obtained from a placental cell bank (e. G., PDAC Bank).

In any of the above embodiments of isolated placental cells, isolated placental cells are generally not differentiated during growth in a growth medium, i.e., a medium prepared to promote proliferation, for example, in a growth medium. In another embodiment, the isolated placental cells do not require a feeder layer to propagate. In another embodiment, the isolated placental cells are cultured in the absence of only a feeder cell layer, which is not differentiated in culture.

In certain embodiments, the isolated placental cells have been perfused to remove blood and remove residual blood; The blood was drained but not perfused to remove residual blood; Or by perfusion of the postpartum placenta in which blood has not been perfused to drain or remove residual blood. In another specific embodiment, said isolated placental cells are obtained by physical and / or enzymatic destruction of placental tissue.

Cell surface markers, molecular markers and genetic markers of placental cells useful in the methods provided herein are described in detail in Section 5.2 below.

In another specific embodiment of the above described method, the isolated placental cells are administered by bolus injection. In another specific embodiment, the isolated placental cells are administered by intravenous infusion. In a specific embodiment, the intravenous infusion is intravenous infusion over about 1 to about 8 hours. In another specific embodiment, said isolated placental cells are administered intracranially. In another specific embodiment, the isolated placental cells are administered intraperitoneally. In another specific embodiment, the isolated placental cells are administered intraarterially. In another specific embodiment of the method of treatment, the isolated placental cells are administered intramuscularly, intradermally, subcutaneously, intrathecally or intravenously.

In another embodiment of the above described method, the isolated placental cells are administered by implantation by surgery into a composition of matter comprising the isolated placental cells. In a specific embodiment, the composition of the material is a matrix or a scaffold. In another specific embodiment, the matrix or scaffold is a hydrogel. In another specific embodiment, the matrix or scaffold is a degassed tissue. In another specific embodiment, the matrix or scaffold is a synthetic biodegradable composition. In another specific embodiment, the matrix or scaffold is a foam.

In another specific embodiment of the method described above, said isolated placental cells are administered once to said subject. In another specific embodiment, the isolated placental cells are administered to the subject in two or more separate administrations. In another specific embodiment, the administration comprises administering about 1 x 10 ⁴ to 1 x 10 ⁵ isolated placental cells, for example placental cells, per kg of the subject. In another specific embodiment, the administration comprises administering about 1 x 10 ⁵ to 1 x 10 ⁶ isolated placental cells per kg of the subject. In another specific embodiment, the administration comprises administering about 1 x 10 ⁶ to 1 x 10 ⁷ isolated placental cells per kg of the subject. In another specific embodiment, the administration comprises administering about 1 x 10 ⁷ to 1 x 10 ⁸ isolated placental cells per kg of the subject. In another specific embodiment, the administration comprises about 1 x 10 ⁶ to about 2 x 10 ⁶ isolated placental cells per kg of the subject; About 2 x 10 ⁶ to about 3 x 10 ⁶ isolated placental cells per kg of said subject; About 3 x 10 ⁶ to about 4 x 10 ⁶ isolated placental cells per kg of said subject; About 4 x 10 ⁶ to about 5 x 10 ⁶ isolated placental cells per kg of said subject; About 5 x 10 ⁶ to about 6 x 10 ⁶ isolated placental cells per kg of said subject; About 6 x 10 ⁶ to about 7 x 10 ⁶ isolated placental cells per kg of said subject; About 7 x 10 ⁶ to about 8 x 10 ⁶ isolated placental cells per kg of said subject; About 8 x 10 ⁶ to about 9 x 10 ⁶ isolated placental cells per kg of said subject; Or about 9 x 10 ⁶ to about 1 x 10 ⁷ isolated placental cells per kg of said subject. In another specific embodiment, the administration comprises administering to the subject about 1 x 10 ⁷ to about 2 x 10 ⁷ isolated placental cells per kg of the subject. In another specific embodiment, the administration comprises administering to the subject about 1.3 x 10 ⁷ to about 1.5 x 10 ⁷ isolated placental cells per kg of the subject. In another specific embodiment, the administration comprises administering to the subject up to about 3 x 10 ⁷ isolated placental cells per kg of the subject. In a specific embodiment, the administration comprises administering about 5 x 10 ⁶ to about 2 x 10 ⁷ isolated placental cells to the subject. In another specific embodiment, the administration comprises administering to the subject about 150 x 10 ⁶ isolated placental cells in a solution of about 20 milliliters.

In a specific embodiment, the administration comprises administering about 5 x 10 ⁶ to about 2 x 10 ⁷ isolated placental cells to the subject, wherein the cells are 10% dextran, such as dextran-40 , 5% human serum albumin, and optionally an immunosuppressant.

In another specific embodiment, the administration comprises intravenously administering about 5 x 10 ⁷ to 3 x 10 ⁹ isolated placental cells. In a specific embodiment, the administration comprises intravenously administering about 9 x 10 ⁸ isolated placental cells or about 1.8 x 10 ⁹ isolated placental cells. In another specific embodiment, the administration comprises administering about 5 x 10 ⁷ to 1 x 10 ⁸ isolated placental cells into a lesion. In another specific embodiment, the administration comprises administering about 9 x 10 ⁷ isolated placental cells into a lesion.

In certain embodiments, the placental cells used in the methods provided herein can be genetically engineered for the production of one or more proteins that promote angiogenesis. In certain embodiments, the protein that promotes angiogenesis is selected from the group consisting of HGF, VEGF (e.g., VEGFD), fibroblast growth factor (FGF) (ANG), epidermal growth factor (EGF), epithelial-neutrophil-activating protein 78 (ENA-78), polystatin, granulocyte colony-stimulating factor (G- , Interleukin-6 (IL-6), IL-8, leptin, monocyte chemoattractant protein-1 (MCP-1), MCP-3, platelet-derived growth factor subunit B (PDGFB), rantes, (TGF-beta1), Trombopoietin (Tpo), Metloproteinase 1 (TIMP1), Tissue Inhibitor of TIMP2, and / or Urokinase Plasminogen Activator Receptor More than one.

3.1 Definitions

As used herein, the term " about "when used in reference to a value mentioned refers to a value within +/- 10% of the stated value.

As used herein, the term "angiogenesis" for a placenta-derived adherent cell described herein means that the cell is capable of forming blood vessel or vascular-like germination, or that the cell is in another cell population, (E. G., The formation of blood vessels or vascular-like structures) in the blood vessels.

As used herein, the term "angiogenesis" refers to an angiogenic process, including, but not limited to, endothelial cell activation, migration, proliferation, matrix reformation, and cell stabilization.

As used herein, the term "derived" means isolated therefrom or otherwise purified. For example, placenta-derived adherent cells are isolated from the placenta. The term "derived" includes cells cultured from tissues, e. G., Cells isolated directly from the placenta, and cells cultured or expanded from the primary isolate.

As used herein, "immunological localization" refers to the use of an immunogenic protein, e. G., An antibody or fragment thereof, in a flow cytometric assay, fluorescence-activated cell sorting, magnetic cell sorting, in situ hybridization, immunohistochemistry, Means detection of a compound, e. G., A cell marker.

As used herein, the term "SH2" refers to an antibody that binds to an epitope on the cell marker CD105. Thus, the cell referred to as SH2 ⁺ is CD105 ⁺ .

As used herein, the terms "SH3" and "SH4" refer to an antibody that binds to an epitope present on the cell marker CD73. Thus, cells referred to as SH3 ⁺ and / or SH4 ⁺ are CD73 ⁺ .

The placenta has a fetal genotype that develops in the placenta, but it also comes into physical contact with maternal tissue during pregnancy. Thus, as used herein, the term "fetal genotype" refers to the genotype of the fetus, for example, the fetus in relation to the placenta-associated fetus in which the specific isolated placental stem cells described herein are obtained, It means genotype. As used herein, the term "maternal genotype" refers to the genotype of the mother that conceives the fetus, for example the fetus associated with the placenta, from which the particular isolated placental stem cells described herein are obtained.

As used herein, the term "isolated cell ", such as" isolated placental cells ", "isolated placental stem cells ", and the like refer to a cell from which the stem cells are derived, ≪ / RTI > For example, at least 50%, 60%, 70% or more of the stem cells exhibiting different marker profiles, such as non-stem cells, or cells that are naturally associated with stem cells during collection and / , 80%, 90%, 95%, or at least 99% of the cells are removed from the stem cells, the cells are "isolated ".

As used herein, the term "versatile" when referring to a cell refers to the ability of the cell to function as a cell of some type of body (not necessarily of all types) or of a cell of some type of body , Or into one or more cells of the three germ layers. In certain embodiments, isolated placental cells (PDACs) as described in Section 5.2 below, which have the ability to differentiate into cells having, for example, neurogenic, cartilage forming and / or osteogenic cells characteristics, are versatile cells.

As used herein, the term "population of isolated cells" refers to a population of cells from which a population of cells is derived, for example, cells substantially separated from other cells of the placenta.

As used herein, the term "placental cell" refers to a primary isolated cell or cultured cell, independent of the number of subcultures after the primary culture, for example, as isolated from the mammalian placenta, Or stem cells or progenitor cells (also referred to herein as "PDACs ") cultured from cells isolated from a mammalian placenta. However, as used herein, the term "placental cells" and placental cells used in the methods provided herein are intended to encompass a variety of mammalian cells, including but not limited to, trophoblast, cytostatic, fusant cell, trophoblast, angiopoietic, embryonic, , Or cells obtained from gonadal bulges of late embryos, such as embryonic germ cells. The placental cells described herein, such as PDACs, are described in pending U.S. Patent Application No. 12 / 622,352, entitled " Amnion Derived Angiogenic Cells "filed on November 19, 2009, the disclosure of which is incorporated herein by reference Lt; RTI ID = 0.0 > adherent < / RTI > A marker or gene expression profile associated with the properties of stem cells, e.g., one or more types of stem cells; Cells are considered "stem cells" if they exhibit the ability to replicate more than 10-40 times in culture, and the ability to differentiate into cells that exhibit the characteristics of at least one of the three different lysozyme layers. Unless otherwise indicated herein, the term "placenta" includes cordages. In certain embodiments, the isolated placental cells disclosed herein are differentiated in vitro, differentiated in vivo, or both under differentiation conditions.

The placental cells used herein are "positive" for this particular marker if the marker is detectable in excess of the background. Detection of a particular marker may be performed, for example, by the use of an antibody, or by an oligonucleotide probe or primer based on the sequence of the gene or mRNA encoding the marker. For example, placental cells are positive for CD73, for example, because CD73 is detectable on placental cells in a detectably large amount (e.g., compared to an isotype control) than in the background. A cell is also positive for such a marker if the marker can be used to distinguish the cell from one or more other cell types or can be used to select or isolate cells when expressed or present by the cell. For example, in the context of antibody-mediated detection, "positive" as indicating the presence of a particular cell surface marker means that the marker is detectable using an antibody specific for the marker, such as a fluorescence-labeled antibody and; "Positive" also refers to a cell that shows a marker in an amount that produces, for example, a signal that detectably exceeds the background in a cell meter. For example, if cells are labeled to enable detection by an antibody specific for CD200, a signal from the antibody control group detectably higher than the signal (e.g., background or isotype control) cells "CD200 ^+" to be. Conversely, "negative" in the same context means that the cell surface marker is not detectable using an antibody specific for such a marker as compared to a control (e.g., background or isotype control). For example, if the cells are not reproducibly detectably labeled with an antibody specific for CD34 to a greater degree than the control (e.g., background or isotype control), the cell is "CD34- ^& quot ;. Markers that are not detected or detectable using antibodies are determined to be positive or negative in a similar manner using a suitable control. For example, when the amount of OCT-4 RNA detected in an RNA from a cell or a population of cells is detectably larger than the background, as determined by RNA detection methods such as RT-PCR, slot blot, etc., OCT-4 ⁺ . ≪ / RTI > Unless otherwise indicated herein, differentiation population ("CD") markers are detected using antibodies. In certain embodiments, when OCT-4 is detectable using RT-PCR, OCT-4 is determined to be present and the cell is "OCT-4 ^{+ &} quot ;.

As used herein, the term "low" when referring to the expression of a detectable marker in flow cytometry refers to whether the marker is expressed by less than 10% of the cells to be tested or by marker in the flow cytometry, It means that one fluorescence is less than 1 log compared to the background.

As used herein, "treating" includes correcting, ameliorating, moderating, or reducing the time course of a disease, disorder or condition, or any of its parameters or symptoms.

4. Brief description of drawing
Figure 1 shows the secretion of selected angiogenic proteins by placenta-derived adherent cells.
Figure 2 shows the angiogenic effect of placenta-derived adherent cell conditioned media on human endothelial cell (HUVEC) tube formation.
Figure 3 shows the angiogenic effect of placenta-derived adherent cell conditioned media on human endothelial cell migration.
Figure 4 shows the effect of placenta-derived adherent cell-conditioned medium on human endothelial cell proliferation.
Figure 5 shows tube formation of HUVEC and placenta-derived adherent cells.
Figure 6 shows the secretion of VEGF and IL-8 by placenta-derived adherent cells under hypoxic and normoxic conditions.
Figure 7 shows the positive effect of PDAC on angiogenesis in a chick chorionic villi angiogenesis model. bFGF: Basic fibroblast growth factor (positive control). MDAMB231: angiogenic breast cancer cell line (positive control). Y axis: degree of angiogenesis.
Figure 8 shows the positive effect of PDAC-conditioned medium (supernatant) on angiogenesis in a chick chorionic villi angiogenesis model. bFGF: Basic fibroblast growth factor (positive control). MDAMB231: angiogenic breast cancer cell line (positive control). Y axis: degree of angiogenesis.
Figure 9: Culture of astrocytes or hydrogen peroxide-producing reactive oxygen species present in co-cultures of astrocytes and PDAC. RFU ROS activity: Relative fluorescence unit for reactive oxygen species.

5. Detailed Description

5.1 Treatment of diseases or conditions related to or resulting from angiogenesis, poor blood flow

Provided herein are methods of treating circulatory diseases or disorders comprising administering an effective amount of a tissue culture platelet-adherent placental cell, such as PDAC, as described in Section 5.2 below. In certain embodiments, the PDAC is angiogenesis. PDACs can support the growth of endothelial and endothelial cell populations, and epithelial and epithelial cell populations, both in vitro and in vivo.

5.1.1 Circulatory diseases

The PDACs provided herein, a population of such cells, and a population of cells comprising PDACs can be used to treat individuals exhibiting various disease states or conditions that would benefit from increased angiogenesis. Examples of such disease states or conditions include myocardial infarction, congestive heart failure, peripheral arterial disease, left ventricular hypoplasia syndrome, diabetic ulcer, bedsore ulcer, venous ulcer, arterial ulcer, burn, nonunion fracture, osteoarthritis, . In certain embodiments, a PDAC and a population of such cells can be used in an individual exhibiting traumatic tissue loss to promote angiogenesis or to prevent scar formation, or in an entire joint replacement or dental prosthesis.

In a more specific embodiment, the PDACs and populations of cells provided herein can be used to treat individuals with a circulatory system failure, e. G., Peripheral vascular disease or individuals with coronary artery disease.

In one aspect, there is provided herein a method of treating a patient with a heart disease or disorder, comprising administering a therapeutic cell composition to a patient having a disease or disorder of the heart or circulatory system and evaluating the patient for an improvement in cardiac function , Wherein said cell composition comprises a PDAC as described herein. In one embodiment, the heart disease is cardiomyopathy. In a specific embodiment, cardiomyopathy is idiopathic, or the cause is known cardiomyopathy. In another specific embodiment, cardiomyopathy is ischemic or non-ischemic in nature. In another embodiment, the disease of the cardiac or circulatory system is selected from the group consisting of angioplasty, aneurysm, angina pectoris, aortic stenosis, aortitis, arrhythmia, arteriosclerosis, arteritis, asymmetric heavy wall hypertrophy (ASH), atherosclerosis, And congestive heart failure (congenital heart defects), congestive heart failure, cardiomyopathy, cardiac hypertrophy, cardiomyopathy, carotid artery disease, congestive heart failure, congestive heart failure, bacterial endocarditis, Barlow syndrome (mitral valve prolapse), bradycardia Cardiomyopathy, heart failure, hypertension, hypotension, idiopathic infant arterial calcification, Kawasaki disease (mucocutaneous lymphadenopathy syndrome), coronary artery disease, coronary artery disease, Eisenmenger syndrome, embolism, endocarditis, skin irritation, , Mucosal skin nodal disease, infantile multiple arteritis), metabolic syndrome, microvascular angina, myocardial infarction (heart attack), myocarditis, paroxysmal atrial (Pulmonary stenosis), Raynaud's disease, renal artery stenosis, renal artery stenosis, neovascular glaucoma, diabetic nephropathy, diabetic angiopathy, phlebitis, pulmonary artery stenosis (pulmonary stenosis) Atherosclerosis, valvular heart disease, varicose ulceration, varicose veins, vasculitis, diabetic nephropathy, diabetic nephropathy, vascular hypertension, rheumatic heart disease, septal defect, asymptomatic ischemia, syndrome X, tachycardia, Ventricular septal defect, Wolf-Parkinson-White syndrome, or endomyopelvic defect.

In another embodiment, the disease of the cardiac or circulatory system is selected from the group consisting of acute rheumatic fever, acute rheumatic endocarditis, acute rheumatic endocarditis, acute rheumatic myocarditis, chronic rheumatic heart disease, mitral valve disease, mitral stenosis, rheumatic mitral regurgitation, (Acute pulmonary heart disease, pulmonary embolism, chronic pulmonary heart disease), posterior scoliosis cardiac disease, myocarditis, endocarditis, cardiomyopathy, cardiomyopathy, Myocardial degeneration, diseases of the circulatory system such as cerebrovascular disease, occlusion and stenosis of brain artery, occlusion of cerebral artery, arterioles, arterioles and capillary diseases (Atherosclerosis, aneurysms), or diseases of the veins and lymphatic vessels.

In another embodiment, the treatment comprises treatment of a subject with cardiomyopathy using a therapeutic cell composition comprising PDAC with or without another cell type. In certain embodiments, an individual is selected for treatment from the treatment, for example, from the ability of cells to support the growth of stem cells or progenitor cells present in other cells, e.g., the heart, from tissue growth or angiogenesis of the tissue, Although they experience benefits from the presence of cellular factors, chemokines, cytokines, etc., cells do not integrate or multiply within the patient. In another embodiment, the patient benefits from a healing treatment with the cell, but the cell does not survive in the patient for an extended period of time. In one embodiment, the cell is progressively attenuated in number, viability or biochemical activity, and in other embodiments there may be a period of activity prior to attenuation of the cell, for example, a period of growth, division or biochemical activity. In another embodiment, aged, non-viable or even dead cells can have beneficial therapeutic effects.

Improvement of a circulatory system disorder or an individual with a disorder wherein the PDAC or therapeutic composition provided herein is administered to a subject can be assessed or attested by a detectable improvement in one or more symptoms of the disease or disorder of the circulatory system.

In another embodiment, an improvement in a disease or disorder entity in the circulatory system (wherein the subject is administered a PDAC, or a therapeutic composition comprising a PDAC) is administered to a subject prior to administration of the PDAC, (CO), cardiac index (CI), pulmonary artery wedge pressure (PAWP) and heart index (CI),% fractionation shortening (% FS), extrusion coefficient (EF) Left ventricular ejection fraction (LVEF); LVEDD, left ventricular end-diastolic diameter (LVESD), contractility (eg, dP / dt), pressure-volume loop, heart work measurement, increased atrial or ventricular function; Increased pumping efficiency, reduced pumping efficiency loss rate, reduced hemodynamic loss of function; And a reduction in complications associated with cardiomyopathy. ≪ Desc / Clms Page number 2 >

Improvements in the subject receiving a therapeutic composition comprising the PDAC, or PDAC provided herein, can also be assessed by a subjective measure, e. G., A self-assessment of the individual's health status after administration.

In certain embodiments, the success of administration of the cells is not based on survival in the individual of the administered PDAC. Instead, success is based on one or more measures of improvement in cardiac or circulatory health, as described above. Thus, the cells do not need to be integrated into the patient's heart or into the blood vessels to beat.

In certain embodiments, the therapeutic methods provided herein can be used to treat PDACs along the mesenchymal pathway, such as toward myocardial production, angiogenesis, or angiogenic phenotype, or into muscle cells, myocardial cells, endothelial cells, Vascular endothelial cells, smooth muscle cells (e. G., Vascular smooth muscle cells).

Administration of PDAC, or a therapeutic composition comprising such a cell, to an individual in need thereof can be effected, for example, by transplantation, insertion (e.g., of the cell itself or of a cell as part of a matrix- For example, directly to the ischemic site in the heart of a subject having a myocardial infarction), infusion, delivery via a catheter, or any other < RTI ID = 0.0 > Can be achieved by means.

In one embodiment, the therapeutic cell composition is provided by injection into an individual in need thereof, e.g., from one to more than one site. In a specific embodiment, the therapeutic cell composition is provided, for example, by intracardiac injection into an ischemic area in the heart. In another specific embodiment, the cells are injected onto a surface of the heart, to an adjacent region, or even to a farther region. In a preferred embodiment, the cells can be directed to the diseased or injured area.

The PDAC may be administered to a subject having a disease or condition of the coronary artery or vascular system at any time when the cell is therapeutically beneficial. In certain embodiments, for example, the PDACs or therapeutic compositions of the present invention are administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23 or 24 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. Administration within a time zone close to myocardial infarction, such as 1-3 or 1-7 days, is more preferable for administration of myocardial infarction at a remote time point, for example, 3 or 7 days later. In another embodiment, the cell or therapeutic composition of the present invention is administered to a patient at an age of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days.

Kits for use in the treatment of myocardial infarction are also provided herein. Kits provide therapeutic cell compositions, including PDACs, and application devices, together with instructions for use, that may be prepared in a pharmaceutically acceptable form, for example, by mixing with a pharmaceutically acceptable carrier. Ideally, the kit may be used in the field, for example in a clinic, or by a first aid provider, to be applied to patients diagnosed with a myocardial infarction or similar cardiac event.

In a specific embodiment of the method of treatment provided herein, the PDAC may be a stem cell (i.e., a stem cell other than a PDAC), a myoblast, an myocyte, a myocyte, a myocyte or a myoblast, an myocyte, a myocyte and / Are administered together with progenitor cells.

In a specific embodiment, the therapeutic methods provided herein comprise administering a therapeutic composition comprising a PDAC, e. G., Said cell, to a patient having a disease of the heart or circulatory system; And assessing the patient for improvement of cardiac function, wherein the therapeutic cell composition is administered as a matrix-cell complex. In certain embodiments, the matrix is a scaffold comprising at least said cell, preferably a bioabsorbable scaffold.

To this end, a population of PDACs that have been contacted (e.g. incubated or cultured in the presence thereof) with one or more factors that stimulate stem or progenitor cell differentiation along cardiogenic, angiogenic, angiogenic or angiogenic pathways are further described herein to provide. These factors are currently known or later determined to stimulate, for example, differentiation of stem cells, along with growth factors, chemokines, cytokines, cellular products, demethylating agents, and cardiogenic, angiogenic, angiogenic or angiogenic pathways or systems. Other factors such as, but not limited to, other factors.

In certain embodiments, the PDACs comprise at least one of a demethylating agent, BMP (bone morphogenic protein), FGF (fibroblast growth factor), Wnt factor protein, hedgehog protein and / or anti-Wnt factor in vitro May be differentiated by cardiogenic, angiogenic, angiogenic or angiogenic pathways or lines by culturing the cells in the presence of factors.

The inclusion of demethylating agents tends to promote PDAC differentiation towards the myocardial production pathway along the mesenchymal line. Differentiation may be achieved by expression of at least one of, for example, cardiac myosin, skeletal myosin, or GATA4 (e. G., Measured by immunostaining or RT-PCR); Or by obtaining a spontaneous or otherwise induced beating rhythm; Or at least partially into the heart muscle of a patient without inducing arrhythmias. The demethylating agents that can be used to initiate this differentiation include 5-azacytidine, 5-aza-2'-deoxycytidine, dimethylsulfoxide, keleritrine chloride, retinoic acid or its salts, But are not limited to, 4- (ethylthio) butyric acid, procainamide, and procaine.

In certain embodiments of the disclosure, the PDAC in contact with the identified one or more factors can be myocardial production, angiogenesis, angiogenic or angiogenic cells, or progenitor cells. Preferably, at least a portion of the cells may be incorporated, at least in part, into the cardiovascular system of the recipient, including, but not limited to, cardiac muscle of the heart, blood vessels and other structures, heart or peripheral blood vessels, In certain embodiments, a differentiated PDAC differentiates into a cell that exhibits more than one characteristic of a myocyte-producing cell or precursor cell thereof, and may be partially or fully integrated into the heart or vascular system of the recipient. In a specific embodiment, the cell does not increase upon administration to the subject an arrhythmia, a heart defect, a vascular defect, or other abnormalities in the circulatory system or health of the individual. In certain embodiments, the PDAC is a stem cell that is naturally present in a patient's cardiac muscle, blood vessel, blood, etc., which self-differentiates, for example, into myocardial cells, or at least along myocardial production, angiogenesis, angiogenesis, And the like.

The PDAC and the population of such cells may be provided therapeutically or prophylactically to a subject having a disease, disorder or condition that has or affects an individual, e. G., A disease or disorder or condition of the heart or circulatory system. Such diseases, disorders or conditions may include atherosclerosis, cardiomyopathy, or cardiac damage, such as ischemic injury, e.g., myocardial infarction or a congestive heart failure due to a wound (acute or chronic).

In certain embodiments, the subject is administered a population of cells comprising a therapeutically effective amount of PDAC, such as PDAC. In a specific embodiment, the population comprises about 50% PDAC. In another specific embodiment, the population is a substantially homogeneous population of PDACs. In another embodiment the population is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% 75%, 80%, 85%, 90%, 95%, 98% or 99% PDAC.

The PDAC may be administered to a subject in an amount sufficient to induce said cell and another therapeutic agent such as insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor VEGF), hepatocyte growth factor (HGF), interleukin 18 (IL-8), antithrombogenic agents (such as heparin, heparin derivatives, urokinase or PPack (dextrophenylalanine proline arginine chloromethyl ketone) (Eg, erythropoietin, erythropoietin, erythropoietin, erythropoietin, erythropoietin, erythropoietin, erythropoietin, erythropoietin, erythropoietin, IGF-I, IGF-II, hepatocyte growth factor (HGF) or a caspase inhibitor), an anti-inflammatory agent (e. G., P38 MAP kinase inhibitor , star , An IL-6 inhibitor, an IL-I inhibitor, a feminolast, a tranilast, a remicade, a sylolimus and / or a non- steroidal anti- inflammatory compound (e.g., acetylsalicylic acid, ibuprofen, Such as, for example, cyclosporin, tacrolimus, mTOR inhibitors such as sylolimus or everolimus; antiproliferatives such as azathioprine, And / or mycophenolate mofetil, a corticosteroid such as prednisolone or hydrocortisone, an antibody such as a monoclonal anti-IL-2Ra receptor antibody, a bacillic ischemia, a daclizumma, a polyclonal anti- Antibodies, such as anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG), and / or monoclonal anti-T cell antibody OKT3, or U.S. Pat. No. 7,468,276 and U.S. patent application publication no. Placental stem cells such as those described in U.S. Patent Application No. 275362, each of which is incorporated herein by reference in its entirety), and / or antioxidants (e.g., probucol; Such as vitamin A, C and / or E, coenzyme Q-10, glutathione, L cysteine, N-acetylcysteine, or any of the antioxidant derivatives, analogs or salts of the foregoing) have. In certain embodiments, a therapeutic composition comprising a PDAC further comprises one or more additional cell types, such as adult cells (e. G., Fibroblast or endodermal cells), stem cells and / or progenitor cells. Such therapeutic agents and / or one or more additional types of cells may be administered to the individual in need thereof, or a combination of two or more such compounds or agents.

In certain embodiments, the subject to be treated is a mammal. In a specific embodiment, the subject to be treated is a human. In a specific embodiment, the subject is a domestic animal or a pet animal. In another specific embodiment, the subject to be treated is a horse, a sheep, a cow, or a bull, a pig, a dog, or a cat.

5.1.2 Treatment of Ischemic Diseases

In certain embodiments, provided herein are methods of treating such individuals, including, for example, administering a therapeutically effective amount of PDAC to a subject having a destruction of blood flow in the peripheral vascular system. In certain specific embodiments, ischemia is peripheral artery disease (PAD), such as severe ischemic ischemia (CLI). In another specific embodiment, ischemia is peripheral vascular disease (PVD), peripheral arterial disease, ischemic vascular disease, ischemic heart disease or ischemic nephropathy.

In a specific embodiment, said bloodstream breakdown is severe ischemia. In another more specific embodiment, the CLI is a severe occlusion of the lower limb arteries, which significantly reduces blood flow. In another more specific embodiment, the CLI is administered to a patient in need of such treatment as ischemic stabilization pain, severe pain of the legs and feet during the absence of an individual, non-healing pressure ulcers on the foot or leg, pain or numbness of the foot, And characterized by dry skin, enlargement of the toenails, absence or reduction of the pulse of the legs or feet, open bed sores, unhealed skin infections or ulcers, and / or dry necrosis of the legs or feet (dry black skin). In another specific embodiment, an individual with CLI experiences loss of one or more fingers and / or an entire limb. In another specific embodiment of the method, the therapeutically effective amount is an amount effective to reduce the loss of limb function and / or oxygen deprivation (hypoxia / anoxia) due to blood flow breakdown in the peripheral vascular system of a subject, The number of PDACs causing the delay of progression of one or more symptoms thereof. In another specific embodiment, the therapeutically effective amount of the isolated PDAC is administered to the subject in order to reduce or eliminate tissue damage caused, for example, by secondary or subsequent bloodstream breaks in or around the limb after the bloodstream breakdown, .

In another embodiment, PDACs may be used for the treatment of stroke, e. G., Ischemic stroke, for example, in the treatment of stroke due to angiogenesis promotion in the ischemic area of the CNS. In one aspect, there is provided a method of treating a subject suffering from a bloodstream destruction in or around an individual's brain, for example, a subject's brain or a subject suffering from a symptom or neurological deficit resulting from blood flow in or around the central nervous system (CNS) There is provided herein a method of treating such an individual, comprising administering an effective amount of an isolated tissue culture platelet-adhering human placenta cell, wherein said isolated placental cell has the characteristics of a pluripotent or stem cell. In certain embodiments, destruction of blood flow causes anoxic or hypoxic damage to the brain or CNS of an individual. As contemplated herein, the treatment of a symptom or neurological deficit in an individual due to blood flow destruction in or around the brain of an individual may result in reperfusion injury, which may be accompanied by bloodstream rupture in or around the brain of such an individual Lt; RTI ID = 0.0 > neurological < / RTI > deficiency.

The placental cells provided herein (e. G., PDACs as described below) are neuroprotective besides angiogenic. In certain embodiments, placental cells are neuroprotective under hypoxic conditions, e.g., under hypoxic conditions (e.g., less than about 5% O ₂ ). In certain embodiments, the placental stem cells are contacted with neurons or other neurons, or astrocytes, such as neurons, neurons, and neurons, as evidenced by increased neurite length in an in vitro co-culture of PDACs and neurons Promotes the health of cells or astrocytes. In another specific embodiment, the PDAC reduces the concentration of one or more reactive oxygen species in a hypoxic environment. In addition, in certain embodiments, the placental cells (e. G., PDAC) comprise at least one of neurotrophic cytokines BDNF, VEGF, HGF, G-CSF, nerve growth factor (NGF) and / or neurotrophin- . Thus, the PDAC may be useful in the treatment of ischemic injury to both the CNS and the PNS, e.g., ischemic injury to the nervous system in the CNS due to stroke, or ischemic injury to the nervous system in the PNS due to gastrointestinal limb ischemia or peripheral vascular disease It is useful for treatment. In another embodiment, the PDAC can be used to treat, for example, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and / or peripheral neuropathy, for example diabetic neuropathy.

5.2 Isolated placental cells and isolated placental cell population

Isolated placental cells useful in the treatment of individuals having a circulatory disease or disorder, such as PDACs, can be obtained from a placenta or a part thereof, which is attached to a tissue culture substrate and has the characteristics of a pluripotent or stem cell, Cell. In certain embodiments, isolated placental cells useful in the methods disclosed herein have the ability to differentiate into non-placental cell types.

The isolated placental cells useful in the methods disclosed herein may be of fetal or maternal origin (i. E. May have a genotype of the fetal or maternal, respectively). Preferably, the isolated placental cells and the group of isolated placental cells are of fetal origin. As used herein, the phrase "fetal origin" or "non-maternal origin" is intended to indicate that a group of isolated placental cells or isolated placental cells is derived from an embryonic or placental construct associated with the fetus, . As used herein, the phrase "maternal origin" indicates that a cell or group of cells is derived from a placental construct associated with the host, e. A population of cells, including isolated placental cells, such as PDACs, or isolated placental cells, may contain isolated placental cells whose origin is the fetal or maternal parent alone, or may contain isolated fetal and maternal origin Lt; RTI ID = 0.0 > placental < / RTI > Isolated placental cells, and isolated placental cells can be identified and selected by morphological characteristics, marker characteristics, and culture characteristics discussed below. In certain embodiments, any placental cell, e. G., A placental stem cell or placenta pluripotent cell described herein, is autologous to a recipient, e. G., A subject having a disease or disorder of the circulatory system. In certain specific embodiments, any placental cell, such as a placental stem cell or placental pluripotent cell described herein, is heterologous to a recipient, e. G., A subject having a disease or disorder of the circulatory system.

5.2.1 Physical and morphological features

The isolated placental cells (PDACs) described herein can be used in tissue culture plates, for example tissue culture plates (e.g., in tissue culture plastics, or extracellular matrices or ligands) when cultured in primary cultures or cell cultures Such as laminin, collagen (e.g., natural or modified collagen), gelatin, fibronectin, ornithine, bitronectin, and extracellular membrane proteins (e.g., Matrigel, BD Discovery Labware, The isolated placenta cells in the culture generally exhibit a fibroblast-like stellate appearance, and a large number of cytoplasmic protrusions extend from the central cell body. However, the isolated placental cells Because the cells exhibit a greater number of such protrusions than the fibroblasts, the cells undergo morphological changes from fibroblasts cultured under the same conditions As it is distinguishable. Morphologically, the isolated placental cells are also possible distinct from hematopoietic stem cells, hematopoietic stem cells, represents a more rounded form or in the form of gravel during culture in general.

In certain embodiments, the isolated placental cells useful in the methods disclosed herein, when cultured in a growth medium, develop embryo-analogs. Embryo-analogs are discontinuous clusters of cells that can grow on the adherent layer of isolated proliferating placental cells. The term " embryo-like "is used because clusters of cells growing from a culture of embryonic stem cells are similar to embryoid bodies. Growth media that can develop in proliferating cultures of placental cells from which embryo-analogs have been isolated include, for example, DMEM-LG (e. G., From Gibco); 2% fetal bovine serum (e.g., from Hyclone Labs.); 1x insulin-transferrin-selenium (ITS); 1x linoleic acid-bovine serum albumin (LA-BSA); 10 ^-9 M dexamethasone (e.g., from Sigma); 10 ^-4 M ascorbic acid 2-phosphate (e.g., from Sigma); Epidermal growth factor 10 ng / mL (from, for example, R & D Systems); And 10 ng / mL of platelet-derived growth factor (PDGF-BB) (from, for example, R & D Systems).

5.2.2 Cell surface, molecular and genetic markers

Isolated placental cells, such as isolated pluripotent placental cells or isolated placental stem cells, and a population of such isolated placental cells useful in the methods disclosed herein, for example, in a method of treating a disease or disorder of the circulatory system, Adherent human placental cells that have the characteristics of stem cells and express a plurality of markers that can be used to identify and / or isolate populations of cells comprising cells or stem cells. In certain embodiments, the PDAC is angiogenic, e.g., in vitro or in vivo. The isolated placental cells and placental cell populations described herein (i.e., two or more isolated placental cells) can be selected from the group consisting of placental cells and placental cells obtained directly from the placenta or any portion thereof (e.g., chorion, placenta, etc.) Containing cell population. The population of isolated placental cells also includes a population of isolated placental cells (i.e., two or more) in the culture, and a population, e.g., a population in a bag. Isolated placental cells described herein are not bone marrow-derived mesenchymal cells, fat-derived mesenchymal stem cells, or mesenchymal cells obtained from umbilical cord blood, placental blood or peripheral blood. Placental cells useful in the methods and compositions described herein, such as placental pluripotent cells and placental cells, are described, for example, in U.S. Patent Nos. 7,311,904; 7,311, 905; And 7,468,276; And United States Patent Application Publication No. 2007/0275362, the disclosures of which are incorporated herein by reference in their entirety.

In certain embodiments, the isolated placental cells are isolated placental stem cells. In another particular embodiment, the isolated placental cells are isolated placental pluripotent cells. In one embodiment, the isolated placental cells, for instance CD34 PDAC is the time of detection by flow cytometry ^- a CD10 ⁺ and CD105 ^+. In another specific embodiment, the isolated CD34 ^-, CD10 ^+, CD105 ⁺ placental cells has the potential to differentiate into a cell and / or cartilage formation phenotype of cells, osteogenic phenotypes of neural cell phenotype. In another specific embodiment, the isolated CD34 ^-, CD10 ^+, CD105 ⁺ placental cells are CD200 ⁺ additionally. In another specific embodiment, the isolated CD34 ^- , CD10 ⁺ , CD105 ⁺ placental cells are further CD45 ^- or CD90 ⁺ . In another specific embodiment, the isolated CD34 ^- , CD10 ⁺ , CD105 ⁺ placental cells are further CD45 ^- and CD90 ⁺ upon detection by flow cytometry. In another specific embodiment, the isolated CD34 ^- , CD10 ⁺ , CD105 ⁺ , CD200 ⁺ placental cells are further CD90 ⁺ or CD45 ^- when detected by flow cytometry. In another specific embodiment, the isolated CD34 ^- , CD10 ⁺ , CD105 ⁺ , CD200 ⁺ placental cells are further CD90 ⁺ and CD45 ^- when detected by flow cytometry, ie the cells are CD34 ^- , CD10 ⁺ , CD45 ^- , CD90 ⁺ , CD105 ⁺ and CD200 ⁺ . In another specific embodiment, the CD34 ^-, CD10 ^+, CD45 ^-, CD90 ^+, CD105 ^+, CD200 ⁺ cells are added to the CD80 ^- and CD86 ^- a.

In another embodiment, the placental cells, when detected by a flow cytometry CD34 ^-, CD10 ^+, CD105 ⁺ and CD200 ^+, and ^{CD38 -, CD45 -, CD80 -} , CD86 -, CD133 -, HLA-DR, DP ^{, DQ -, SSEA3 -, SSEA4} -, CD29 +, CD44 +, CD73 +, CD90 +, CD105 +, HLA-A, B, C +, PDL1 +, ABC-p +, and / or OCT-4 ⁺ of More than one. In another embodiment, the disclosed random CD34 ^-, CD10 ^+, CD105 ⁺ CD29 ⁺ cells in addition, CD38 ^-, CD44 ⁺ a, at least one of CD54 ^+, SH3 ⁺ or SH4 ^+. In another specific embodiment, the cell is further CD44 ⁺ . Wherein any of the isolated CD34 ^-, from CD10 ^+, another specific embodiment of the CD105 ⁺ placental cells, the cells are added to the ^{CD117 -, CD133 -, KDR -} (VEGFR2 -), HLA-A, B, C +, HLA -DP, DQ, DR ^-, or a programmed death ligand -1 (PDL1) ⁺ one or more, or a combination of any of the.

In other ^{embodiments, CD34 -, CD10 +, CD105} + cells are added to the ^{CD13 +, CD29 +, CD33 +} , CD38 -, CD44 +, CD45 -, CD54 +, CD62E -, CD62L -, CD62P -, SH3 + ^{(CD73 +), SH4 + (} CD73 +), CD80 -, CD86 -, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD117 -, CD144 / VE- cadherin ^low, CD184 / CXCR4 ^-, CD200 ^{+, CD133 -, OCT-4} +, SSEA3 -, SSEA4 -, ABC-p +, KDR - (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ, DR -, HLA-G - , Or a programmed killing-1 ligand (PDL1) ⁺ , or any combination thereof. In other ^{embodiments, CD34 -, CD10 +, CD105} + cells are added to the ^{CD13 +, CD29 +, CD33 +} , CD38 -, CD44 +, CD45 -, CD54 / ICAM +, CD62E -, CD62L -, CD62P -, ^{SH3 + (CD73 +), SH4} + (CD73 +), CD80 -, CD86 -, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD117 -, CD144 / VE- cadherin ^me, CD184 / CXCR4 ^{- , CD200 +, CD133 -, OCT} -4 +, SSEA3 -, SSEA4 -, ABC-p +, KDR - (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ, DR -, HLA- G ^-, and the programmed death ligand -1 (PDL1) ^+.

In another specific embodiment, any of the placental cells described herein is ABC-p ⁺ when detected by flow cytometry or OCT-4 ⁺ (POU5F1 ⁺ ) when determined by RT-PCR, wherein ABC-p is (Also known as breast cancer resistant protein (BCRP) and mitoxantrone resistant protein (MXR)) and OCT-4 is an octamer-4 protein (POU5F1). In another specific embodiment, when any of the placental cells described herein are determined by the additional flow cytometry to SSEA3 ^- or SSEA4 ^-, wherein SSEA3 is a stage-specific embryonic antigen-3, SSEA4 is phase-specific embryonic antigen 4 to be. In another specific embodiment, any of the placental cells described herein are added to SSEA3 ^- and SSEA4 ^- a.

In another specific embodiment, any of the placental cells described herein, MHC-I ⁺ (e.g., HLA-A, B, C +), MHC-II - ( for example, HLA-DP, DQ, DR ^- at least one ^-) or HLA-G. In another specific embodiment, any of the placental cells described herein, MHC-I ⁺ (e.g., HLA-A, B, C +), MHC-II - ( for example, HLA-DP, DQ, DR ^-), and HLA-G ^- it is at least one.

A population of isolated placental cells, or a population of cells, such as a population of placental cells, enriched, for example, for such cells, comprising isolated placental cells useful in the methods and compositions disclosed herein are also provided herein do. For example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 80%, 85%, 90%, 95% or 98% of isolated CD10 ⁺ , CD105 ⁺ and CD34 ^- placental cells; That is, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% A population of cells comprising isolated placental cells that are CD10 ⁺ , CD105 ⁺ and CD34 ^- placental cells isolated from 80%, 85%, 90%, 95% or 98% In a specific embodiment, the isolated CD34 ^-, CD10 ^+, CD105 ⁺ placental cells are CD200 ⁺ additionally. In another specific embodiment, the isolated CD34 ^- , CD10 ⁺ , CD105 ⁺ , CD200 ⁺ placental cells are further CD90 ⁺ or CD45 ^- when detected by flow cytometry. In another specific embodiment, the isolated CD34 ^-, CD10 ^+, CD105 ^+, CD200 ⁺ placental cells are CD90 ⁺ and CD45 when adding detected by flow cytometry ^as-is. In another specific embodiment, the above-described any of the isolated CD34 ^-, CD10 ^+, CD105 ⁺ placental cells are CD29 ^+, CD38 additionally ^- at least one, CD44 ^+, CD54 ^+, SH3 ⁺ or SH4 ^+. In another specific embodiment, the isolated CD34 ^-, CD10 ^+, CD105 ⁺ placental cells, and isolating a CD34 ^-, CD10 ^+, CD105 ^+, CD200 ⁺ placental cells are CD44 ⁺ in addition. Wherein the isolated CD34 ^-, CD10 ^+, CD105 ⁺ in specific embodiments of any of the cell populations containing placental cells, the isolated placental cells are more CD13 ^+, CD29 ^+, CD33 ^+, CD38 as ^-, CD44 ^+, CD45 ^{- , CD54 +, CD62E -, CD62L} -, CD62P -, SH3 + (CD73 +), SH4 + (CD73 +), CD80 -, CD86 -, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD117 - , CD144 / VE- cadherin ^{low, CD184 / CXCR4 -, CD200 +} , CD133 -, OCT-4 +, SSEA3 -, SSEA4 -, ABC-p +, KDR - (VEGFR2 -), HLA-A, B, C ^+, HLA-DP, DQ, DR ^- a, or programmed death ligand -1 (PDL1) ⁺ one or more of, or a any combination of ^-, HLA-G. In another specific ^{embodiment, CD34 -, CD10 +, CD105} + cells are added to the ^{CD13 +, CD29 +, CD33 +} , CD38 -, CD44 +, CD45 -, CD54 / ICAM +, CD62E -, CD62L -, CD62P - ^{, SH3 + (CD73 +),} SH4 + (CD73 +), CD80 -, CD86 -, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD117 -, CD144 / VE- cadherin ^low, CD184 / CXCR4 ^{-, CD200 +, CD133 -,} OCT-4 +, SSEA3 -, SSEA4 -, ABC-p +, KDR - (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ, DR -, HLA -G < ^- & gt ^; , and the programmed death-1 ligand (PDL1) ⁺ .

In certain embodiments, useful the isolated placental cells in the methods and compositions described herein are CD10 ^+, CD29 ^+, CD34 ^-, CD38 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ^+, SH4 ⁺ , SSEA3 ^-, SSEA4 ^-, and OCT-4 ⁺ and ABC-p ⁺ one or more or all of, wherein the isolated placental cells are obtained by physical and / or enzymatic destruction of the placental tissue. In a specific embodiment, the isolated placental cells are OCT-4 ⁺ and ABC-p ⁺ . In another specific embodiment, the isolated placental cells are OCT-4 ⁺ and CD34 ^-, wherein said isolated placental cells have at least one of the following characteristics: CD10 ^+, CD29 ^+, CD44 ^+, CD45 ^-, CD54 ⁺ , CD90 ⁺ , SH3 ⁺ , SH4 ⁺ , SSEA3 ^- and SSEA4 ^- . In another specific embodiment, the isolated placental cells are OCT-4 ^+, CD34 ^-, CD10 ^+, CD29 ^+, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH3 ^+, SH4 ^+, SSEA3 ^- and SSEA4 ^- a . In another embodiment, the isolated placental cells are ^{OCT-4 +, CD34 -,} SSEA3 - and SSEA4 ^- a. In another specific embodiment, the isolated placental cells are OCT-4 ⁺ and CD34 ^- , and SH2 ⁺ or SH3 ⁺ . In another specific embodiment, the isolated placental cells are OCT-4 ^+, CD34 ^- a, and SH2 ⁺ SH3 ^+. In another specific embodiment, the isolated placental cells are ^{OCT-4 +, CD34 -,} SSEA3 -, and SSEA4 ^- is a SH2 or SH3 ^{+ +.} In another specific embodiment, the isolated placental cells are OCT-4 ⁺ and CD34 ^-, and SH2 ⁺ or SH3 ⁺ and, CD10 ^+, CD29 ^+, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SSEA3 ^-, or SSEA4 ^- is one or more of. In another specific embodiment, the isolated placental cells are OCT-4 ^+, CD34 ^-, CD10 ^+, CD29 ^+, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SSEA3 ^- and SSEA4 ^-, and SH2 ⁺ or SH3 ⁺ to be.

In another embodiment, the isolated placental cells useful in the methods and compositions disclosed herein are SH2 ⁺ , SH3 ⁺ , SH4 ^+, and OCT-4 ⁺ . In another specific embodiment, the isolated placental cells are ^{CD10 +, CD29 +, CD44 +} , CD54 +, CD90 +, CD34 - a ^-, CD45 ^-, SSEA3 ^-, or SSEA4. In another embodiment, the isolated placental cells are SH2 ⁺ , SH3 ⁺ , SH4 ⁺ , SSEA3 ^- and SSEA4 ^- . In another specific embodiment, the isolated placental cells are SH2 ^+, SH3 ^+, SH4 ^+, SSEA3 ^- and SSEA4 ^-, CD10 ^+, CD29 ^+, CD44 ^+, CD54 ^+, CD90 ^+, OCT-4 ^+, CD34 ^- or CD45 ^- .

In another embodiment, useful for the isolated placental cells in the methods and compositions disclosed herein are CD10 ^+, CD29 ^+, CD34 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ^+, and SH4 ⁺ and ; Wherein the isolated placental cells are added to the OCT-4 ^+, SSEA3 ^- or SSEA4 ^- at least one.

In certain embodiments, the isolated placental cells useful in the methods and compositions disclosed herein are CD200 ⁺ or HLA-G ^- . In a specific embodiment, the isolated placental cells are CD200 ⁺ and HLA-G ^- . In another specific embodiment, the isolated placental cells are further CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the isolated placental cells are added to the CD34 ^-, CD38 ^-, or CD45 ^- a. In another specific embodiment, the isolated placental cells are added to the CD34 ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, the stem cells are CD34 ^- , CD38 ^- , CD45 ^- , CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said isolated CD200 ⁺ or HLA-G ^- placental cells promote the formation of embryo-analogs in a population of placental cells comprising isolated placental cells under conditions permitting the formation of embryo-analogs . In another specific embodiment, the isolated placental cells are isolated from placental cells that are not stem or pluripotent cells. In another specific embodiment, said isolated placental cells are isolated from placental cells that do not display these markers.

In another embodiment, the population of cells useful in the methods and compositions described herein is a population of cells enriched, e. G., Comprising, CD200 ⁺ , HLA-G ^- stem cells. In a specific embodiment, the population is a population of placental cells. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cells in the cell population are isolated CD200 ⁺ , HLA-G ^- Placental cells. Preferably, at least about 70% of the cells in the cell population are isolated CD200 ⁺ , HLA-G ^- placental cells. More preferably, at least about 90%, 95% or 99% of the cells are isolated CD200 ⁺ , HLA-G ^- placental cells. In a specific embodiment of the cell population, said isolated CD200 ⁺ , HLA-G ^- placental cells are also CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said isolated CD200 ⁺ , HLA-G ^- placental cells are also CD34 ^- , CD38 ^- or CD45 ^- . In another specific embodiment, said isolated CD200 ^+, HLA-G ^- placental cells are also CD34 ^-, CD38 ^-, CD45 ^-, CD73 ⁺ and CD105 ⁺ a. In another embodiment, the cell population produces one or more embryo-analogs when cultured under conditions permitting the formation of embryo-analogs. In another specific embodiment, the cell population is isolated from placental cells, not stem cells. In another specific embodiment, said isolated CD200 ⁺ , HLA-G ^- placental cells are isolated from placental cells that do not exhibit such markers.

In another embodiment, the isolated placental cells useful in the methods and compositions described herein are CD73 ⁺ , CD105 ^+, and CD200 ⁺ . In another specific embodiment, the isolated placental cell is HLA-G ^- . In another specific embodiment, the isolated placental cells are CD34 ^-, CD38 ^-, or CD45 ^- a. In another specific embodiment, the isolated placental cells are CD34 ^- , CD38 ^- and CD45 ^- . In another specific embodiment, the isolated placental cells are CD34 ^-, CD38 ^-, CD45 ^- a ^-, and HLA-G. In another specific embodiment, the isolated CD73 ⁺ , CD105 ⁺ and CD200 ⁺ placental cells are cultured under conditions that allow the formation of embryo-analogs of a population of placental cells, including isolated placental cells, Promoting the formation of embryo-analogs. In another specific embodiment, the isolated placental cells are isolated from placental cells that are not isolated placental cells. In another specific embodiment, isolated placental cells are isolated from placental cells that do not exhibit such markers.

In another embodiment, a population of cells useful in the methods and compositions of treatment described herein is a population of cells enriched for, for example, such cells, including isolated CD73 ⁺ , CD105 ⁺ , CD200 ⁺ placental cells. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cell population comprises isolated CD73 ⁺ , CD105 ⁺ CD200 ⁺ placental cells. In another embodiment, at least about 70% of the cells in the population of cells are isolated CD73 ⁺ , CD105 ⁺ , CD200 ⁺ placental cells. In another embodiment, at least about 90%, 95%, or 99% of the cells in the population of cells are isolated CD73 ⁺ , CD105 ⁺ , CD200 ⁺ placental cells. In a specific embodiment of this population, the isolated placental cells are HLA-G ^- . In another specific embodiment, the isolated placental cells are added to the CD34 ^-, CD38 ^-, or CD45 ^- a. In another specific embodiment, the isolated placental cells are added to the CD34 ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, the isolated placental cells are added to the CD34 ^-, CD38 ^-, CD45 ^- a ^- and HLA-G. In another specific embodiment, the population of cells produces one or more embryo-analogs when cultured under conditions permitting the formation of embryo-analogs. In another specific embodiment, the population of placental cells is isolated from placental cells, not stem cells. In another specific embodiment, the population of placental cells is isolated from placental cells that do not display these markers.

In certain other embodiments, the isolated placental cells are CD10 ^+, CD29 ^+, CD34 ^-, CD38 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ^+, SH4 ^+, SSEA3 ^-, SSEA4 ^{-, OCT-4 +, HLA-G} - is at least one or ABC-p ^+. In a specific embodiment, the isolated placental cells are CD10 ^+, CD29 ^+, CD34 ^-, CD38 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ^+, SH4 ^+, SSEA3 ^-, SSEA4 ^-, and OCT-4 ⁺ . In another specific embodiment, the isolated placental cells are ^{CD10 +, CD29 +, CD34 -} , CD38 -, CD45 -, the ^{CD54 +, SH2 +, SH3 +} , SH4 ⁺ and. In another specific embodiment, the isolated placental cells are ^{CD10 +, CD29 +, CD34 -} , CD38 -, CD45 -, the ^{CD54 +, SH2 +, SH3 +} , SH4 + and OCT-4 ^+. In another specific embodiment, the isolated placental cells are CD10 ^+, CD29 ^+, CD34 ^-, CD38 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, HLA-G ^-, SH2 ^+, an SH3 ^+, SH4 ⁺ . In another specific embodiment, the isolated placental cells are OCT-4 ⁺ and ABC-p ⁺ . In another specific embodiment, the isolated placental cells are SH2 ⁺ , SH3 ⁺ , SH4 ⁺ and OCT-4 ⁺ . In another embodiment, the isolated placental cells are ^{OCT-4 +, CD34 -,} SSEA3 -, and SSEA4 ^- a. In a specific embodiment, said isolated ^{OCT-4 +, CD34 -,} SSEA3 - and SSEA4 ^- placental cells are more CD10 ^+, CD29 ^+, CD34 as ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ⁺ and SH4 ⁺ . In another embodiment, the placental cells isolated are OCT-4 ⁺ and CD34 ^- , and SH3 ⁺ or SH4 ⁺ . In another embodiment, the isolated placental cells are CD34 ^-, and CD10 ^+, the ^{CD29 +, CD44 +, CD54 +} , CD90 + or OCT-4 ^+.

In another embodiment, the isolated placental cells useful in the methods and compositions described herein are CD200 ⁺ and OCT-4 ⁺ . In a specific embodiment, the isolated placental cells are CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the isolated placental cell is HLA-G ^- . In another specific embodiment, said isolated CD200 ⁺ , OCT-4 ⁺ placental cells are CD34 ^- , CD38 ^- or CD45 ^- . In another specific embodiment, said isolated CD200 ⁺ , OCT-4 ⁺ placental cells are CD34 ^- , CD38 ^- and CD45 ^- . In another specific embodiment, said isolated CD200 ^+, OCT-4 ⁺ placental cells are CD34 ^-, CD38 ^-, CD45 ^- a ^-, CD73 ^+, CD105 ^+, and HLA-G. In another specific embodiment, the isolated CD200 ⁺ , OCT-4 ⁺ placental cells are cultured under conditions that allow for the formation of embryo-analogs when a population of placental cells comprising isolated cells is incubated with one or more embryos - promotes the production of analogs. In another specific embodiment, said isolated CD200 ⁺ , OCT-4 ⁺ placental cells are isolated from placental cells other than stem cells. In another specific embodiment, said isolated CD200 ⁺ , OCT-4 ⁺ placental cells are isolated from placental cells that do not exhibit these characteristics.

In another embodiment, the population of cells useful in the methods and compositions described herein is a population of cells enriched for example, such as CD200 ⁺ , OCT-4 ⁺ placental cells. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cells in the cell population are isolated CD200 ⁺ , OCT-4 ⁺ Placental cells. In another embodiment, at least about 70% of the cells are the isolated CD200 ⁺ , OCT-4 ⁺ placental cells. In another embodiment, at least about 80%, 90%, 95%, or 99% of the cells in the population of cells are the isolated CD200 ⁺ , OCT-4 ⁺ placental cells. In a specific embodiment of the isolated population, said isolated CD200 ⁺ , OCT-4 ⁺ placental cells are further CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said isolated CD200 ⁺ , OCT-4 ⁺ placental cells are additionally HLA-G ^- . In another specific embodiment, said isolated CD200 ^+, OCT-4 ⁺ placental cells are added to the CD34 ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, said isolated CD200 ^+, OCT-4 ⁺ placental cells are added to the CD34 ^-, CD38 ^-, CD45 ^- a ^-, CD73 ^+, CD105 ^+, and HLA-G. In another specific embodiment, the population of cells produces one or more embryo-analogs when cultured under conditions permitting the formation of embryo-analogs. In another specific embodiment, the population of cells is isolated from placental cells that are not CD200 ⁺ , OCT-4 ⁺ placental cells isolated. In another specific embodiment, the population of cells is isolated from placental cells that do not exhibit these markers.

In another embodiment, the isolated placental cells useful in the methods and compositions described herein are CD73 ⁺ , CD105 ⁺ and HLA-G ^- . In another specific embodiment, the isolated CD73 ^+, CD105 ^+, and HLA-G ^- placental cells are added to the CD34 ^-, CD38 ^-, or CD45 ^- a. In another specific embodiment, the isolated ^{CD73 +, CD105 +, HLA-} G - placental cells are added to the CD34 ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, the isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells are additionally OCT-4 ⁺ . In another specific embodiment, the isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells are further CD200 ⁺ . In another specific embodiment, the isolated ^{CD73 +, CD105 +, HLA-} G - placental cells are additionally CD34 ^-, CD38 ^-, CD45 ^-, an OCT-4 ⁺ and CD200 ^+. In another specific embodiment, isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells are cultured in a population of placental cells containing the cells under conditions permitting the formation of embryo-analogs, Promoting the formation of analogs. In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells are isolated from placental cells other than isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells. In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells are isolated from placental cells that do not display these markers.

In another embodiment, the population of cells useful in the methods and compositions described herein is a population of cells enriched for, for example, such cells, including isolated CD73 ⁺ , CD105 ⁺ and HLA-G ^- placental cells. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cells in the population of cells have isolated CD73 ⁺ , CD105 ⁺ , And HLA-G ^- placental cells. In another embodiment, at least about 70% of the cells in the population of cells are isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells. In another embodiment, at least about 90%, 95% or 99% of the cells in the population of cells are isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells. In a specific embodiment of said populations, said isolated ^{CD73 +, CD105 +, HLA-} G - placental cells are added to the CD34 ^-, CD38 ^-, or CD45 ^- a. In another specific embodiment, said isolated ^{CD73 +, CD105 +, HLA-} G - placental cells are added to the CD34 ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells are additionally OCT-4 ⁺ . In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells are further CD200 ⁺ . In another specific embodiment, said isolated ^{CD73 +, CD105 +, HLA-} G - placental cells are added to the CD34 ^-, CD38 ^-, CD45 ^-, an OCT-4 ⁺ and CD200 ^+. In another specific embodiment, the population of cells is isolated from placental cells other than CD73 ⁺ , CD105 ⁺ , HLA-G ^- placental cells. In another specific embodiment, the population of cells is isolated from placental cells that do not exhibit these markers.

In another embodiment, the isolated placental cells useful in the methods and compositions described herein are CD73 ⁺ and CD105 ⁺ , and the population of isolated placental cells comprising the CD73 ⁺ , CD105 ⁺ cells allows the formation of embryo-analogs Lt; RTI ID = 0.0 > embryo-like < / RTI > In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ placental cells are further CD34 ^- , CD38 ^- or CD45 ^- . In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ placental cells are further CD34 ^- , CD38 ^- and CD45 ^- . In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ placental cells are further OCT-4 ⁺ . In another specific embodiment, it said isolated CD73 ^+, CD105 ⁺ placental cells are OCT-4 ^+, CD34 additionally ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ placental cells are isolated from placental cells other than said cells. In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ placental cells are isolated from placental cells that do not exhibit these characteristics.

In another embodiment, the population of cells useful in the methods and compositions described herein is a population of cells enriched for, for example, such cells, including isolated placental cells that are CD73 ⁺ , CD105 ⁺ Promotes the formation of one or more embryo-analogs in the population when isolated populations of placental cells are cultured under conditions permitting the formation of embryo-analogs. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cells in the cell population are isolated from the isolated CD73 ⁺ , CD105 ⁺ Placental cells. In another embodiment, at least about 70% of the cells in the population of cells are the isolated CD73 ⁺ , CD105 ⁺ placental cells. In another embodiment, at least about 90%, 95%, or 99% of the cells in the population of cells are the isolated CD73 ⁺ , CD105 ⁺ placental cells. In a specific embodiment of said populations, said isolated CD73 ^+, CD105 ⁺ placental cells are added to the CD34 ^-, CD38 ^-, or CD45 ^- a. In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ placental cells are further CD34 ^- , CD38 ^- and CD45 ^- . In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ placental cells are further OCT-4 ⁺ . In another specific embodiment, said isolated CD73 ⁺ , CD105 ⁺ placental cells are further CD200 ⁺ . In another specific embodiment, it said isolated CD73 ^+, CD105 ⁺ placental cells are added to the CD34 ^-, CD38 ^-, CD45 ^-, an OCT-4 ⁺ and CD200 ^+. In another specific embodiment, the population of cells is isolated from placental cells other than the isolated CD73 ⁺ , CD105 ⁺ placental cells. In another specific embodiment, the population of cells is isolated from placental cells that do not exhibit these markers.

In another embodiment, the isolated placental cells useful in the methods and compositions described herein are OCT-4 ⁺ , and when the population of isolated placental cells comprising the cells is cultured under conditions permitting the formation of embryo-analogs Promoting the formation of one or more embryo-analogs in the population. In a specific embodiment, said isolated OCT-4 ⁺ placental cells are further CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the isolated placental cells are OCT-4 ⁺ the added by CD34 ^-, CD38 ^-, or CD45 ^- a. In another specific embodiment, said isolated OCT-4 ⁺ placental cells are further CD200 ⁺ . In another specific embodiment, it said the isolated OCT-4 ⁺ placental cells are ^{CD73 +, CD105 +, CD200 +} , CD34 additionally ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, said isolated OCT-4 ⁺ placental cells are isolated from placental cells that are not OCT-4 ⁺ placental cells. In another specific embodiment, said isolated OCT-4 ⁺ placental cells are isolated from placental cells that do not exhibit these characteristics.

In another embodiment, a population of cells useful in the methods and compositions described herein is a population of cells enriched for example, such as OCT-4 ⁺ isolated placental cells, Lt; RTI ID = 0.0 > embryo-analog < / RTI > in the population when cultured under conditions that allow formation of embryo-analogs. In various embodiments, at least about 10% of the population of the cells, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells are said isolated OCT-4 ⁺ Placental cells. In another embodiment, at least about 70% of the cells in the population of cells are the isolated OCT-4 ⁺ placental cells. In another embodiment, at least about 80%, 90%, 95%, or 99% of the cells in the population of cells are the isolated OCT-4 ⁺ placental cells. In a specific embodiment of said populations, the isolated placental cells are OCT-4 ⁺ the added by CD34 ^-, CD38 ^-, or CD45 ^- a. In another specific embodiment, the isolated placental cells are OCT-4 ⁺ the added by CD34 ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, said isolated OCT-4 ⁺ placental cells are further CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said isolated OCT-4 ⁺ placental cells are further CD200 ⁺ . In another specific embodiment, it said the isolated OCT-4 ⁺ placental cells are ^{CD73 +, CD105 +, CD200 +} , CD34 additionally ^-, CD38 ^- and CD45 ^- a. In another specific embodiment, said population of cells is isolated from placental cells other than said cells. In another specific embodiment, the population of cells is isolated from placental cells that do not exhibit these markers.

In another embodiment, the isolated placental cells useful in the methods and compositions described herein is an isolated ^{HLA-A, B, C +} , CD45 -, CD133 - and CD34 ^- cells, the placenta. In another embodiment, a population of cells useful in the methods and compositions described herein is a population of cells comprising isolated placental cells, wherein at least about 70%, at least about 80%, at least about 90% %, at least about 95% or at least about 99% of the cells are ^{HLA-a, B, C +} , CD45 isolated ^- a placental ^cell-, CD133 ^- and CD34. In a specific embodiment, the population of the cells of the placenta, or being isolated placental cells are ^{HLA-A, B, C +} , CD45 - are isolated from the placenta non-placental cells ^-, CD133 ^- and CD34. In another specific embodiment, said isolated placental cells are non-maternal origin. In another specific embodiment, the population of isolated placental cells is substantially free of maternal components, for example at least about 40%, 45%, 50%, 55%, 60% %, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of the cells are of non-maternal origin.

In another embodiment, the isolated placental cells useful in the methods and compositions described herein is an isolated ^{CD10 +, CD13 +, CD33 +} , CD45 -, CD117 - CD133 and ^- the placenta cells. In another embodiment, a population of cells useful in the methods and compositions described herein is a population of cells comprising isolated placental cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 70% About 95% or at least about 99% of the cells are the isolated CD10 ⁺ , CD13 ⁺ , CD33 ⁺ , CD45 ^- , CD117 ^- and CD133 ^- placental cells. In a specific embodiment, said isolated placental cell, or a population of isolated placental cells, is isolated from placental cells other than said isolated placental cells. In another specific embodiment, it said isolated ^{CD10 +, CD13 +, CD33 +} , CD45 -, CD117 - and ^{CD133-placental} stem cells are non-maternal origin, and, that have the fetal genotype. In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90% 95%, 98% or 99% of the cells are of non-maternal origin. In another specific embodiment, said isolated placental cells, or a population of isolated placental cells, are isolated from placental cells that do not exhibit these characteristics.

In another embodiment, the isolated placental cells useful in the methods and compositions described herein is an isolated CD10 ^-, CD33 ^-, CD44 ^+, CD45 ^- and CD117 ^- the placenta cells. In another embodiment, a population of cells useful in the methods and compositions described herein is a population of cells enriched for, for example, such cells, including isolated placental cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of the isolated cells are CD10 ^-, CD33 ^-, CD44 ^+, CD45 ^-, CD117, and ^- the placenta cells. In a specific embodiment, said isolated placental cells, or a population of isolated placental cells, are isolated from placental cells other than said cells. In another specific embodiment, said isolated placental cells are non-maternal origin. In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90% 98% or 99% of said cells are of non-mature origin. In another specific embodiment, said isolated placental cells, or a population of isolated placental cells, are isolated from placental cells that do not exhibit these markers.

In another embodiment, the isolated placental cells, useful in the methods and compositions described herein are isolated ^{CD10 -, CD13 -, CD33 -} , CD45 - and CD117 ^- the placenta cells. Of the enrichment for containing placental cells, e.g., those cells ^- In another embodiment, useful cell population in the methods and compositions described herein is an isolated CD10 ^-, CD13 ^-, CD33 ^-, CD45 ^- and CD117 group, wherein at least about 70% in the group, at least about 80%, at least about 90%, at least about 95% or at least about 99% of the cells are ^{CD10 -, CD13 -, CD33 -} , CD45 - and CD117 ^- placental cells to be. In a specific embodiment, said isolated placental cells, or a population of isolated placental cells, are isolated from placental cells other than said cells. In another specific embodiment, said isolated placental cells are non-maternal origin. In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90% 98% or 99% of said cells are of non-mature origin. In another specific embodiment, said isolated placental cells, or a population of isolated placental cells, are isolated from placental cells that do not exhibit these characteristics.

In another embodiment, useful for the isolated placental cells in the methods and compositions described herein are HLA A, B, C ^+, CD45 ^-, CD34 ^- and CD133 ^- and, more CD10 ^+, CD13 ^+, CD38 ^+, with CD44 ^{+ , CD90 +, CD105 +, CD200} + and / or HLA-G ^-, and is / or CD117 negative. In another embodiment, a population of cells useful in the methods described herein is a population of cells comprising isolated placental cells, wherein at least about 20%, 25%, 30%, 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or about 99% cells, HLA a, B, C ^-, CD45 ^-, CD34 ^-, CD133 ^-, and is added to the CD10, CD13, CD38, CD44, CD90, CD105, CD200-positive, and / or CD117 and / or HLA-G negative in the isolated placental cells. In a specific embodiment, said isolated placental cells, or a population of isolated placental cells, are isolated from placental cells other than said cells. In another specific embodiment, said isolated placental cells are non-maternal origin. In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90% 98% or 99% of said cells are of non-mature origin. In another specific embodiment, said isolated placental cells, or a population of isolated placental cells, are isolated from placental cells that do not exhibit these markers.

In another embodiment, useful for the isolated placental cells in the methods and compositions described herein are as determined by antibody binding CD200 ⁺ and CD10 ^+, upon antibody binding and determined by both RT-PCR CD117 ^- of the isolated placenta It is a cell. In another embodiment, the isolated placental cells useful in the methods and compositions described herein, CD10 ^+, CD29 ^-, CD54 ^+, CD200 ^+, HLA-G ^-, MHC class I ⁺ and β-2- microglobulin ⁺ Isolated placental cells, for example placental stem cells or placental pluripotent cells. In another embodiment, the isolated placental cells useful in the methods and compositions described herein are placental cells, wherein expression of at least one of the cell markers is less than that of mesenchymal stem cells (e. G., Bone marrow-derived mesenchymal stem cells) At least twice as high. In another specific embodiment, said isolated placental cells are non-maternal origin. In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90% 98% or 99% of said cells are of non-mature origin.

In another embodiment, useful for the isolated placental cells in the methods and compositions described herein, CD10 ^+, CD29 ^+, CD44 ^+, CD45 ^-, CD54 / ICAM ^+, CD62E ^-, CD62L ^-, CD62P ^-, CD80 ^-, CD86 ^{-, CD103 -, CD104 -,} CD105 +, CD106 / VCAM +, CD144 / VE- cadherin ^low, CD184 / CXCR4 ^-, β2- microglobulin ^low, MHC-I ^{me, MHC-II -, HLA-} G and ^low- an / or PDL1 the isolated placental cells, at least one of ^{the low,} for example, placental stem cells or placental pluripotent cells. In a specific embodiment, the isolated placental cells are at least CD29 ⁺ and CD54 ⁺ . In another specific embodiment, the isolated placental cells are at least CD44 ⁺ and CD106 ⁺ . In another specific embodiment, the isolated placental cells are at least CD29 ⁺ .

In another embodiment, a population of cells useful in the methods and compositions described herein comprises isolated placental cells and comprises at least 50%, 60%, 70%, 80%, 90%, 95% 98% or 99% ^{CD10 +, CD29 +, CD44 +} , CD45 -, CD54 / ICAM +, CD62-E -, CD62-L -, CD62-P -, CD80 -, CD86 -, CD103 -, CD104 -, ^{CD105 +, CD106 / VCAM +,} CD144 / VE- cadherin ^about, CD184 / CXCR4 ^-, β2- microglobulin ^about, HLA-I ^about, HLA-II ^-, HLA-G is isolated more than one ^drug and / or PDL1 ^about Placental cells. In another specific embodiment, the cells, at least 50% in the group, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells were CD10 ^+, CD29 ^+, CD44 ^+, CD45 ^{-, CD54 / ICAM +, CD62-E} -, CD62-L -, CD62-P -, CD80 -, CD86 -, CD103 -, CD104 -, CD105 +, CD106 / VCAM +, CD144 / VE- cadherin ^about, CD184 / CXCR4 ^-, β2- microglobulin ^about, MHC-I ^about, MHC-II ^-, the HLA-G ^{approximately,} and ^about PDL1.

In another embodiment, useful for the isolated placental cells in the methods and compositions described herein, CD10 ^+, CD29 ^+, CD34 ^-, CD38 ^-, CD44 ^+, CD45 ^-, CD54 ^+, CD90 ^+, SH2 ^+, SH3 ^+, SH4 ^+, SSEA3 ^-, SSEA4 ^-, OCT-4 ^+, and an ABC-p ⁺ one or more or the ex-wife was isolated placental cells of, where ABC-p is a placenta-specific ABC transporter protein (breast cancer resistant protein (BCRP) And also known as mitoxantrone-resistant protein (MXR)), and the isolated placental cells are obtained by perfusion of a mammal, e.g., human placenta, perfused to remove cord blood and remove residual blood.

In another specific embodiment of any of the above characteristics, the expression of a cell marker (e.g., a differentiation cluster or an immunogenic marker) is determined by flow cytometry; In another specific embodiment, the expression of the marker is determined by RT-PCR.

In gene profiling, it is confirmed that isolated placental cells, and a population of isolated placental cells, are distinct from other cells, such as mesenchymal stem cells, such as bone marrow-derived mesenchymal stem cells. Based on the expression of one or more genes in which the expression of the gene is significantly higher in isolated placental cells or in isolated umbilical cord stem cells than in bone marrow-derived mesenchymal stem cells, Lt; RTI ID = 0.0 > mesenchymal stem cells. ≪ / RTI > In particular, the isolated placental cells useful in the methods of treatment provided herein are significantly higher (i. E., At least 2-fold < RTI ID = 0.0 > For example, bone marrow-derived mesenchymal stem cells, wherein one or more genes are selected from the group consisting of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, IL11, IL11, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA- LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, ZC3H12A or any combination of these genes. See, for example, U.S. Patent Application Publication No. 2007/0275362, the disclosure of which is incorporated herein by reference in its entirety. In certain embodiments, the expression of the one or more genes is determined by, for example, RT-PCR or microarray analysis using, for example, a U133-A microarray (apimetrix). In another specific embodiment, said isolated placental cells are selected from the group consisting of DMEM-LG (e. G., From Gibco); 2% fetal bovine serum (e.g., from Hyclone Labs.); 1x insulin-transferrin-selenium (ITS); 1x linoleic acid-bovine serum albumin (LA-BSA); 10 ^-9 M dexamethasone (e.g., from Sigma); 10 ^-4 M ascorbic acid 2-phosphate (e.g., from Sigma); Epidermal growth factor 10 ng / mL (from, for example, R & D Systems); And from 10 ng / mL of platelet-derived growth factor (PDGF-BB) (from, for example, R & D Systems), for example from about 3 to about 35 population doublings And expressing the at least one gene. In another specific embodiment, the isolated placental cell-specific or isolated umbilical cell-specific gene is CD200.

Specific sequences for these genes are listed in Table 1 under the names of GenBank accession numbers NM_001615 (ACTG2), BC065545 (ADARB1), NM_181847 (AMIGO2), AY358590 (ARTS-1), BC074884 (B4GALT6), BC008396 (BCHE), BC020196 (DS2), AF338241 (ELOVL2), AY336105 (F2RL1), NM_018215 (DS1), BC031103 (CD200), NM_001845 (COL4A1), NM_001846 (COL4A2), BC052289 (CPA4), BC094758 (DMD), AF293359 (IL16), AY416799 (GATA6), BC075798 (GPR126), NM_016235 (GPRC5B), AF340038 (ICAM1), BC000844 (IER3), BC066339 (IGFBP7), BC013142 (IL1A), BT019749 (IL6), BC007461 ), BC060825 (LIPG), BC065240 (LRAP), BC010444 (MATN2), BC011908 (MEST), BC068455 (NFE2L3), NM_014840 (NUAK1), AB006755 (PCDH7), NM_014476 (PDLIM3), BC126199 (SEQ ID NO: 2), BC090862 (RTN1), BC002538 (SERPINB9), BC023312 (ST3GAL6), BC001201 (ST6GALNAC5), BC126160 or BC065328 (SLC12A8), BC025697 (TCF21), BC096235 (TGFB2), BC005046 ).

In a specific embodiment, the isolated placental cells are selected from the group consisting of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A, respectively, detectably higher levels than the same number of bone marrow-derived mesenchymal stem cells when cultured under equivalent conditions.

In a specific embodiment, the placental cell is selected from the group consisting of CD200 and ARTS1 (an aminopeptidase modulator of type 1 tumor necrosis factor); ARTS-1 and LRAP (white blood cell-derived arginine aminopeptidase); IL6 (interleukin-6) and TGFB2 (transforming growth factor, beta 2); IL6 and KRT18 (keratin 18); IER3 (extreme early response 3), MEST (mesodermal-specific transcript homolog) and TGFB2; CD200 and IER3; CD200 and IL6; CD200 and KRT18; CD200 and LRAP; CD200 and MEST; CD200 and NFE2L3 (nuclear factor (red blood cell-derived 2) -like 3); Or CD200 and TGFB2 at a detectably higher level than the same number of bone marrow-derived mesenchymal stem cells (BM-MSCs), wherein said bone marrow-derived mesenchymal stem cells are cultured in the presence of the placental stem cells It has undergone the same number of subcultures as the subculture. In another specific embodiment, the placental cells are ARTS-1, CD200, IL6 and LRAP; ARTS-1, IL6, TGFB2, IER3, KRT18 and MEST; CD200, IER3, IL6, KRT18, LRAP, MEST, NFE2L3, and TGFB2; ARTS-1, CD200, IER3, IL6, KRT18, LRAP, MEST, NFE2L3, and TGFB2; Or IER3, MEST and TGFB2 at a detectably higher level than the same number of bone marrow-derived mesenchymal stem cell (BM-MSC) cells, wherein said bone marrow- It has undergone the same number of subcultures as the number of subcultures the cells underwent.

Expression of the above-mentioned genes can be evaluated by standard techniques. For example, probes based on the sequence of the gene (s) can be individually selected and constructed by conventional techniques. For example, a microarray containing probes for one or more of the genes, such as the GENECHIP ® human genome U133A 2.0 array of Apimetrix, or the GeneChip® human genome U133 Plus 2.0 from Affymetrix, Clara). ≪ / RTI > The expression of these genes can be assessed even if the sequence for a particular genbank registry number is modified because probes specific for the modified sequence can be readily generated using well known standard techniques.

The level of expression of these genes can be used to identify the identity of a population of isolated placental cells, to identify a population of cells as containing at least a plurality of isolated placental cells, and the like. A group of isolated placental cells with confirmed identity may be clonal, and may be, for example, a population of isolated placental cells expanded from a single isolated placental cell, for example, an isolated placenta extending from several isolated placental cells A population of cells comprising cells alone, or a group of cells comprising isolated placental cells as described herein and one or more other types of cells.

Expression levels of these genes can be used to select a population of isolated placental cells. For example, if expression of one or more of the genes listed above is significantly higher in a sample from a population of cells than in an equivalent population of mesenchymal stem cells, a population of cells, for example, a clon- . Such a selection may be a selection of populations from a plurality of isolated placental cell populations, populations of a number of cells whose identity is unknown.

The isolated placental cells may be tested for expression levels of the one or more genes, for example, in a mesenchymal stem cell control, based on expression levels of one or more of such genes, for example, May be selected by comparison with the expression level of one or more genes. In one embodiment, the expression level of said one or more genes in a sample comprising the same number of mesenchymal stem cells is used as a control. In another embodiment, the control for the isolated placental cells tested under certain conditions is a value representing the level of expression of said at least one gene in the mesenchymal stem cells under said conditions.

In certain embodiments, placental cells (e. G., PDAC) useful in the methods provided herein do not express CD34 upon detection by immunological localization after 4 to 21 days exposure to 1 to 100 ng / mL VEGF . In a specific embodiment, the placental adherent cells are adherent to tissue culture plastics. In another specific embodiment, the population of cells comprises an angiogenic factor such as vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), platelet derived growth factor (PDGF ) Or basic fibroblast growth factor (bFGF), induce endothelial cells to form shoots or tube-like structures.

In yet another aspect, at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of the cells in the PDAC, cell population, e.g. PDAC population, PDAC-BB, TIMP-1, PDGF-BB, and TIMP- 2, uPAR, or galectin-1, for example, into a culture medium in which cells or cells grow. In another embodiment, PDAC is normal oxygen conditions (e. G., About 20% or about 21% O ₂₎ under the low-oxygen conditions as compared to (e. G., About 5% 0 less than ₂₎ CD202b, IL-8 and / RTI > and / or increased levels of VEGF.

In another embodiment, any of the PDACs described herein, or a population of cells comprising the PDAC, can cause the formation of shoots or tubular-like structures in a population of endothelial cells contacting the placenta-derived adherent cells. In a specific embodiment, the PDACs are co-cultured with human endothelial cells to produce extracellular matrix proteins, e.g. collagen types I and IV, in or on a substrate such as placenta collagen or Matrigel (TM) for at least 4 days, Or growth factor (bFGF) when cultured in the presence of an anti-angiogenic factor and / or an angiogenic factor such as vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), platelet derived growth factor (PDGF) or basic fibroblast growth factor - It forms a similar structure or supports endothelial cell germination. In another embodiment, any population of cells comprising the placenta-derived adherent cells described herein is administered to an angiogenic agent such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF) , Basic fibroblast growth factor (bFGF), or interleukin-8 (IL-8), whereby human endothelial cells secrete extracellular matrix proteins (eg, , For example, to form shoots or tube-like structures when cultured in the presence of collagen types I and IV.

In another embodiment, any such population of cells, including placenta-derived adherent cells (PDAC), secrete angiogenic factors. In a specific embodiment, the cell population is selected from the group consisting of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF) ). In another specific embodiment, a population of cells comprising PDAC secretes one or more angiogenic factors to induce human endothelial cells to migrate in an in vitro wound healing assay. In another specific embodiment, the population of cells comprising placenta-derived adherent cells induces maturation, differentiation or proliferation of human endothelial cells, endothelial precursors, myocytes or myoblasts.

The isolated placental cells described herein can be used in a variety of forms including, for example, DMEM-LG (Gibco), 2% Fetal Bovine Serum (FCS) (Hyclon Laboratories), Ix Insulin-Transferrin-Selenium 10 ^-9 M dexamethasone (Sigma), 10 ^-4 M ascorbic acid 2-phosphate (Sigma), epidermal growth factor (EGF) 10 ng / ml (RAND Systems), platelet derived growth factor (Such as cell surface markers and / or gene expression profiles) in primary culture or in proliferation in medium containing 10 ng / ml of cell surface marker and / or gene expression profile (PDGF-BB) 10 ng / ml (RAND Systems), and 100 U penicillin / 1000 U streptomycin Combination).

In certain embodiments of any placental cells disclosed herein, the cell is a human cell. In certain embodiments of any of the placental cells disclosed herein, the cell marker property or gene expression profile is for a human marker or human gene.

In another specific embodiment of the cell population comprising isolated placental cells, or isolated placental cells, the cells or populations are expanded, for example at least, about or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times, or more, or at least 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, In another specific embodiment of said isolated placental cell, or a population of cells comprising isolated stem cells, said cell or population is a primary isolate. In another specific embodiment of a population of cells comprising isolated placental cells, or isolated placental cells, as disclosed herein, the isolated placental cells are of fetal origin (i. E. Have a fetal genotype).

In certain embodiments, the isolated placental cells do not differentiate during growth in a growth medium, i.e., a medium produced to promote proliferation, such as during growth in a growth medium. In another specific embodiment, the isolated placental cells do not require a feeder layer to propagate. In another specific embodiment, due to the absence of the feeder cell layer alone, the isolated placental cells are not differentiated in culture in the absence of a feeder layer.

In another embodiment, the cells useful in the methods and compositions described herein are isolated placental cells, wherein a plurality of said isolated placental cells are aldehyde dehydrogenase (ALDH) activity assayed by aldehyde dehydrogenase . Such assays are known in the art (see, for example, Bostian and Berts, Biochem. J., 173, 787, (1978)). In a specific embodiment, the ALDH assay uses ALDEFLUOR® (Aldagen, Inc., Ashland, Oreg.) As a marker of aldehyde dehydrogenase activity. In a specific embodiment, the majority is from about 3% to about 25% of the cells in the population of cells. In another embodiment, when assessed by aldehyde dehydrogenase activity assay using aldehyde dehydrogenase activity as an indicator of aldehyde dehydrogenase activity, a number of isolated cord cells are positive for aldehyde dehydrogenase, A collection of umbilical cord cells, e. G., Pluripotent isolated cord cells, is provided herein. In a specific embodiment, the majority is from about 3% to about 25% of the cells in the population of cells. In another embodiment, the population of isolated placental cells or isolated umbilical cord cells is at least 3-fold or more than 5-fold higher than the population of bone marrow-derived mesenchymal stem cells cultured under approximately the same and identical conditions, Activity.

In certain embodiments of any cell population comprising isolated placental cells described herein, placental cells within said cell population are substantially free of cells bearing a maternal genotype; For example, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% 99% have fetal genotypes. In another particular embodiment of any cell population comprising the isolated placental cells described herein, the population of cells comprising the placental cells is substantially free of cells bearing the parental genotype; For example, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% % Have fetal genotype.

In a specific embodiment of any of said isolated placental cells, or a population of cells of isolated placental cells, at least about 95% or about 99% of the karyotypes of said cells, or cells within said population, are normal. In yet another specific embodiment of any of the above placental cells or cell populations, the cells, or cells within a population of cells, are of non-maternal origin.

Isolated placental cells, or groups of isolated placental cells, which possess any of these marker combinations, may be combined in any ratio. Any two or more of said isolated placental cell populations may be combined to form a population of isolated placental cells. For example, a population of isolated placental cells may be divided into a first population of isolated placental cells defined by one of the marker combinations described above, and a second population of isolated placental cells defined by another combination of the markers described above 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70, 40, 60: 50, 60: 40, 70: 30, 80:20, 90:10, 95: 5, 96: 4, 97: 3, 98: 2 or about 99: 1. In a similar manner, any three, four, five or more of the above-described isolated placental cells or a group of isolated placental cells may be combined.

Isolated placental cells useful in the methods and compositions described herein may be obtained by the disruption of, for example, placental tissue, with or without enzymatic digestion (see section 5.3.3) or perfusion (see section 5.3.4) have. For example, perfusing a perfused mammalian placenta with umbilical cord blood removed and residual blood removed; Perfusing the perfusion solution in the placenta; And collecting the perfusion solution, wherein the perfusion solution after perfusion comprises a population of placental cells comprising isolated placental cells; And isolating a plurality of said isolated placental cells from a population of said cells. In a specific embodiment, the perfusion solution is passed through both umbilical vein and umbilical artery and is collected after exuding from the placenta. In another specific embodiment, the perfusion solution is passed through the umbilical vein and collected from the umbilical artery, or through the umbilical artery and collected from the umbilical vein.

In various embodiments, the isolated placental cells contained within a population of cells obtained from the placental perfusion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% At least 99.5%. In another specific embodiment, the isolated placental cells collected by perfusion include fetal and maternal cells. In another specific embodiment, the isolated placental cells collected by perfusion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% fetal cells.

In another specific embodiment, a composition comprising a population of isolated placental cells as described herein, collected by perfusion, is provided herein, wherein said composition is a solution of a perfusion solution used to collect isolated placental cells At least a portion thereof.

The isolated population of isolated placental cells described herein can be obtained by digesting placental tissue with a tissue-destroying enzyme to obtain a population of placental cells comprising such cells, isolating a plurality of placental cells from the remaining placental cells, . ≪ / RTI > The entire placenta or any portion thereof can be digested to obtain the isolated placental cells described herein. For example, in a specific embodiment, the placental tissue may be the entire placenta, amniotic membrane, chorionic membrane, a combination of amniotic membrane and chorionic membrane, or any combination of the foregoing. In another specific embodiment, the tissue destroying enzyme is trypsin or collagenase. In various embodiments, the isolated placental cells contained within a population of cells obtained from placental digestion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5%.

The isolated population of placental cells described above, and the population of isolated placental cells, will generally be at least about 1 x 10 ⁵ , 5 x 10 ⁵ , 1 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ , 5 x 10 ⁸ , 1 x 10 ⁹ , 5 x 10 ⁹ , 1 x 10 ¹⁰ , 5 x 10 ¹⁰ , 1 x 10 ¹¹ or more isolated placental cells have. A population of isolated placental cells useful in the methods of treatment described herein may be isolated by, for example, trypan blue exclusion, for example at least 50%, 55%, 60%, 65%, 70%, 75% %, 85%, 90%, 95%, 98% or 99% of live-isolated placental cells.

5.2.3 Growth of culture

The growth of the isolated placental cells described herein in section 5.4.2 is, in part, dependent on the particular medium selected for growth, as for any mammalian cell. Under optimal conditions, isolated placental cells typically multiply the number within about one day. During incubation, the isolated placental cells described herein adhere to the surface of a substrate at the time of culture, such as a tissue culture container (e.g., tissue culture dish plastic, fibronectin-coated plastic, etc.) to form a monolayer.

A population of placental cells comprising the isolated placental cells described herein can form embryo-analogs when cultured under suitable conditions, i.e., three-dimensional clusters of cells grow at the top of the adherent cell layer. Cells in the embryo-analog express markers associated with extreme early stem cells, such as OCT-4, Nanog, SSEA3 and SSEA4. Typically, cells in embryo-analogs are not adherent to the culture substrate, such as the isolated placental cells described herein, but tend to remain attached to such adherent cells during incubation. Embryo-like cells are dependent on adherent isolated placental cells for viability, since embryo-analogs are not formed in the absence of adherent isolated placental cells. Thus, adherent isolated placental cells promote the growth of one or more embryo-analogs in a population of placental cells comprising adherent isolated placental cells. Without wishing to be bound by theory, it is believed that the cells of the embryo-analog grow on adherent isolated placental cells, such that the embryonic stem cells grow on the feeder cell layer.

5.3 Methods for Obtaining Isolated Placental Cells

5.3.1 Stem cell collection composition

Methods for collecting and isolating placental cells, for example the isolated placental cells described in Section 5.2.2 above, are further provided herein. Generally, such cells are obtained from a mammalian placenta using a physiologically acceptable solution, for example, a cell collection composition. An exemplary cell collection composition is described in detail in US Patent Application Publication No. 2007/0190042, entitled " Improved Medium for Collecting Placental Stem Cells and Preserving Organs ", the disclosure of which is incorporated herein by reference in its entirety .

The cell collection composition may be in any physiologically acceptable solution suitable for the collection and / or culturing of cells, e. G., Isolated placental cells described herein, such as saline solution (e. G., Phosphate- buffered saline, Kreb ), Modified Crab solution, Eagle solution, 0.9% NaCl, etc.), a culture medium (for example, DMEM, H.DMEM, etc.) and the like.

The cell collection composition can be used to prevent the death of the isolated placental cells, to prevent the death of the isolated placental cells, from the time of collection to the time of culture tending to preserve the isolated placental cells, Reducing the number of placental cells isolated in a population of < RTI ID = 0.0 > Such components include, for example, apoptosis inhibitors (e.g., a caspase inhibitor or a JNK inhibitor); (Eg, magnesium sulfate, an antihypertensive drug, an atrial sodium diuretic peptide (ANP), adrenocorticotropin, a corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, Indomethacin or magnesium sulfate, phosphodiesterase inhibitors, etc.); Necrosis inhibitors (e. G., 2- (lH-indol-3-yl) -3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); TNF-a inhibitors; And / or oxygen-carrying perfluorocarbons (e.g., perfluorooctyl bromide, perfluorodecyl bromide, etc.).

The cell collection composition may include one or more tissue-degrading enzymes, such as metalloprotease, serine protease, neutral protease, RNase, or DNase, and the like. Such enzymes include collagenases (e.g., collagenase I, II, III or IV, collagenase from Clostridium histolyticum, etc.); But are not limited to, desipase, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The cell collection composition may comprise a bactericidal effective amount or a bacteriostatic effective amount of an antibiotic. In certain non-limiting embodiments, the antibiotic is selected from the group consisting of macrolides (e.g., tobramycin), cephalosporins (e.g., cephalexin, cephradine, cefuroxime, Erythromycin, penicillin (e.g., penicillin V) or a quinolone (such as oproxacin, ciprofloxacin or norfloxacin), tetracycline, streptomycin, and the like. In certain embodiments, the antibiotic is active against Gram (+) and / or Gram (-) bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus, and the like. In one embodiment, the antibiotic is gentamicin (e.g., about 0.005% to about 0.01% (w / v) in the culture medium).

The cell collection composition may also comprise one or more of the following compounds: adenosine (from about 1 mM to about 50 mM); D-glucose (from about 20 mM to about 100 mM); Magnesium ions (from about 1 mM to about 50 mM); A macromolecule having a molecular weight greater than 20,000 daltons (in one embodiment, present in an amount sufficient to maintain endothelium integrity and cell viability) (e.g., synthetic or naturally occurring colloids, polysaccharides such as dextran or polyethylene Glycols (present from about 25 g / l to about 100 g / l, or from about 40 g / l to about 60 g / l); Antioxidants (e.g., butylated hydroxy anisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E (present at about 25 [mu] M to about 100 [mu] M)); A reducing agent (e.g., N-acetylcysteine (present from about 0.1 mM to about 5 mM)); An agonist (e.g., verapamil (present at about 2 [mu] M to about 25 [mu] M) to prevent calcium entry into the cell; Nitroglycerin (e.g., from about 0.05 g / l to about 0.2 g / l); An anticoagulant (in one embodiment, in an amount sufficient to assist in the prevention of clotting of residual blood) (e.g., heparin or hirudin (present at a concentration of from about 1000 units / l to about 100,000 units / l); Or an amiloride containing compound such as amiloride, ethylisopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride (present at about 1.0 [mu] M to about 5 [mu] M) .

5.3.2 Collection and handling of the placenta

In general, the human placenta is recovered immediately after delivery, just after its onset. In a preferred embodiment, the placenta is recovered after pre-consent from the patient and after taking the patient's full history and relating it to the placenta. Preferably, the history continues after delivery. This history can be used to coordinate subsequent use of the placenta or isolated placental cells harvested therefrom. For example, isolated human placental cells may be used in personalized medicine for parents, siblings or other relatives of infants or infants associated with the placenta in light of their history.

Before recovery of isolated placental cells, umbilical cord blood and placental blood are preferably removed. In certain embodiments, cord blood in the placenta is recovered after delivery. Conventional cord blood collection methods can be applied to the placenta. Typically, a needle or cannula is used to bleed the placenta with the aid of gravity (see, for example, U.S. Patent No. 5,372,581 (Anderson); U.S. Patent No. 5,415,665 (Hessel et al.)). A needle or cannula is typically placed in the umbilical vein and the placenta can be gently massaged to help release cord blood from the placenta. Such umbilical cord blood collection may be performed commercially, for example, from LifeBank USA (Cedar Knoll, New Jersey). Preferably, the placenta is excreted by gravity without further manipulation to minimize tissue disruption during cord blood collection.

Typically, for recovery of umbilical cord blood, for example by perfusion or tissue dissociation, and collection of stem cells, the placenta is transported from the delivery room or delivery room to another location, for example a laboratory. The placenta is preferably transported in a sterile adiabatic transport device (keeping the temperature of the placenta between 20-28 DEG C), for example, the proximal cord is used as a forceps, and the placenta is placed in a sterile, zip-lock plastic bag And placed in an insulating container. In another embodiment, the placenta is transported in a cord blood collection kit as described in substantially pending U.S. Patent No. 7,147,626. Preferably, the placenta is delivered to the laboratory within 4 to 24 hours after delivery. In certain embodiments, the proximal umbilical cords are clamped, preferably within 4-5 cm (centimeters) of the insertion into the placenta disc, prior to cord blood collection. In another embodiment, the proximal cord is clamped after cord blood collection, but before further processing of the placenta.

The placenta may be stored at room temperature or at a temperature between 5 ° C and 25 ° C under sterile conditions prior to cell collection. The placenta may be stored for a period of time ranging from 4 hours to 24 hours, 48 hours or less, or more than 48 hours prior to perfusion of the placenta to remove any residual umbilical cord blood. In one embodiment, the placenta is harvested from about 0 hours to about 2 hours after onset. The placenta is preferably stored in a solution of the anticoagulant at a temperature of from 5 캜 to 25 캜. Suitable anticoagulant solutions are well known in the art. For example, heparin or a solution of sodium warfarin may be used. In a preferred embodiment, the anticoagulant solution comprises a heparin solution (e.g., 1% w / w in a 1: 1000 solution). Placenta cells are collected after the bleeding placenta is preferably stored for less than 36 hours.

Once generally collected and prepared as described above, the mammalian placenta or a portion thereof can be treated in any manner known in the art to obtain isolated placental cells and can be perfused or destroyed, for example, , For example, with one or more tissue-destroying enzymes.

5.3.3 Physical destruction and enzymatic digestion of placental tissue

In one embodiment, the stem cells are collected by physical destruction of part or all of the organ from the mammalian placenta. For example, the placenta or a portion thereof can be squeezed, cut, cut, diced, chopped, macerated, and the like. The tissue can then be cultured to obtain a population of isolated placental cells. Typically, placental tissue is destroyed using, for example, culture media, saline solution or stem cell collection composition (see section 5.5.1 and below).

The placenta can be dissected out as constituents prior to physical destruction and / or enzymatic digestion and stem cell recovery. Isolated placental cells may be obtained from whole placenta, amniotic membrane, chorion, cord, placental lobe, or any combination thereof, in whole or in part. Preferably, the isolated placental cells are obtained from placental tissue comprising amniotic membrane and chorion. Typically, the isolated placental cells are small blocks of placental tissue, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, Or by destruction of blocks of placental tissue that is at least 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or about 1000 cubic millimeters. Any destructive method can be used, provided that the destruction method, when determined, for example, by the triplan blue exclusion, results in a majority, more preferably a majority, more preferably at least 60%, 70% , 80%, 90%, 95%, 98% or 99% are viable.

Generally, isolated adherent placental cells may be collected at any time within the first about 3 days after exposure from the placenta or a portion thereof, but are preferably collected between about 8 hours and about 18 hours after the onset.

In a specific embodiment, the destroyed tissue is cultured in a tissue culture medium suitable for proliferation of isolated placental cells (see, e.g., section 5.6, which describes incubation of placental cells, e. G. PDAC).

In another specific embodiment, the isolated placental cells are harvested by physical destruction of placental tissue, including enzymatic digestion, which can be accomplished by using one or more tissue digestive enzymes. The placenta or a portion thereof may also be physically destroyed and digested with one or more enzymes and then the resulting material may be impregnated into the cell collection composition or mixed into the cell collection composition.

A preferred cell collection composition comprises one or more tissue disruptive enzyme (s). Enzymes that can be used to destroy placental tissue include papain, deoxyribonuclease, serine protease such as trypsin, chymotrypsin, collagenase, dispase or elastase. Serine proteases can be inhibited by alpha 2 microglobulin in serum, and therefore the medium used for digestion is usually serum-free. EDTA and DNase are commonly used in enzymatic digestion procedures to increase the efficiency of cell recovery. The package is preferably diluted to avoid capturing cells in the viscous package.

Any combination of tissue digestive enzymes may be used. Typical concentrations for digestion with trypsin include from 0.1% to about 2% trypsin, for example about 0.25% trypsin. Proteases can be combined, i.e. two or more proteases can be used in the same digestion reaction, or they can be used sequentially to liberate placental cells, such as placental stem cells and placental pluripotent cells. For example, in one embodiment, the placenta or a portion thereof is first digested with a suitable amount of collagenase I of about 1 to about 2 mg / ml, for example, for 30 minutes, followed by incubation with a trypsin at about 0.25% Lt; RTI ID = 0.0 > 37 C < / RTI > for 10 minutes. Serine proteases are preferably used successively following the use of other enzymes.

In another embodiment, a stem cell collection composition comprising stem cells, or a composition comprising a chelator such as ethylene glycol bis (2- Aminoethyl ether) -N, N, N ', N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA).

After digestion, the pellet is washed three times with, for example, a culture medium, and the washed cells are seeded into a culture flask. Cells are then isolated by differential attachment and characterized for, for example, survival rate, cell surface markers, differentiation, and the like.

In the case of a part of the placenta including both the entire placenta or both the fetal and maternal cells (for example, when a part of the placenta contains chorionic villus or placental lobe), the isolated placental cells are separated from both fetal and maternal sources It will be appreciated that a mixture of derived placental cells may be included. In the case of a part of the placenta (e.g. amniotic membrane) containing a number of maternal cells that do not contain or can be neglected, the placental cells isolated therefrom are almost exclusively fetal placental cells (i. E. Placental cells).

Following the differential trypsin treatment (see section 5.3.5 below), culturing in fresh growth medium in one or more new culture vessels (optionally followed by a second differential trypsin treatment step) The placental cells described in .2 can be isolated from the destroyed placental tissue.

5.3.4 Placental perfusion

Placental cells, for example the placental cells described in section 5.2.2 above, can also be obtained by perfusion of the mammalian placenta. Methods of obtaining placental cells by perfusion through the mammalian placenta are described, for example, in U.S. Patent Nos. 7,045,148 and 7,255,729 (Hariri), and U.S. Patent Application Publication Nos. 2007/0275362 and 2007/0190042, Is incorporated herein by reference in its entirety.

For example, placental cells can be harvested by perfusion through the placental vasculature using a cell collection composition as a perfusion solution. In one embodiment, the placenta of the mammal is perfused by passing a perfusion solution through either or both of the umbilical artery and the umbilical vein. The flow of perfusion solution through the placenta can be achieved, for example, using gravity flow into the placenta. Preferably, the perfusion solution is pushed through the placenta using a pump, for example a peristaltic pump. For example, a sterile connection apparatus, such as a cannula connected to a sterile tubing (tubing), for example TEFLON (TEFLON) ^® or plastic cannula can be intubated in umbilical vein. The sterile connection is connected to the perfusion manifold.

In preparation for perfusion, the placenta is preferably placed (for example, hanging) in such a way that the umbilical artery and umbilical vein are located at the highest point of the placenta. The placenta can be perfused by passing a perfusion fluid through the placenta vasculature and surrounding tissues. By passing the perfusion fluid through the umbilical vein and collecting it from the umbilical artery, or by passing the perfusion fluid through the umbilical artery and collecting from the umbilical vein, the placenta can also be perfused.

For example, in one embodiment, the umbilical artery and the umbilical vein are simultaneously connected to a pipette that is connected to a reservoir of perfusion solution, for example, via a flexible connection. The perfusion solution is passed into the umbilical vein and artery. The perfusion solution is collected in a suitable open container from the surface of the placenta attached to the maternal uterus during pregnancy and / or through the walls of the blood vessels into the surrounding tissues of the placenta and / or through these walls. Also, the perfusion solution may be introduced through the umbilical opening and flowed or exuded from the opening in the wall of the placenta that interfaces with the uterine wall of the mother. The placental cells collected by the above method, which can be referred to as the "pan" method, are generally a mixture of fetal and maternal cells.

In another embodiment, the perfusion solution is collected from the umbilical artery through the umbilical vein, or collected from the umbilical vein through the umbilical artery. The placental stem cells collected by the above method, which can be referred to as the "closed circuit" method, are generally almost entirely fetal cells.

It will be appreciated that the perfusion using the pan method, that is, the perfusion that is collected after the perfusion fluid exudates from the maternal side of the placenta, produces a mixture of fetal and maternal cells. As a result, the cells harvested by this method can include placental cells of both fetal and maternal origin, for example, a mixed population of placental stem cells or placenta pluripotent cells. On the other hand, in the perfusion of a closed-circuit method that only passes through the placenta vasculature, where the perfusion fluid passes through one or two placental vessels and is collected only through the remaining vessel (s), a group of fetal placental cells is almost exclusively collected do.

In one embodiment, a closed circuit perfusion method may be performed as follows. The postpartum placenta was obtained within about 48 hours after birth. Pick up the umbilical cord with a forceps and cut it on a forceps. The umbilical cord can be discarded or processed to recover the umbilical cord stem cells, for example, and / or to process the umbilical membrane for the production of biomaterials. The amniotic membrane may be maintained during perfusion or may be detached from the chorioamnion, for example using non-dissection peeling with a finger. If the amniotic membrane is separated from the choroid before perfusion, it can be discarded or processed, for example, to obtain stem cells by digestion with, for example, enzymes, or to obtain stem cells, for example amniotic membrane biomaterials, For example, in U.S. Patent Application Publication No. 2004/0048796, the disclosure of which is incorporated herein by reference in its entirety. All visible blood clots and residual blood are cleaned in the placenta using, for example, sterile gauze, and then the umbilical blood vessels are exposed by, for example, partially cutting the umbilical membrane to expose the umbilical section. The blood vessels are identified and opened, for example by closing the crocodile gripper forward through the cut end of each blood vessel. A device such as a plastic tubing connected to a peristaltic or peristaltic pump is then inserted into each placental artery. The pump may be any pump suitable for the purpose, for example a peristaltic pump. A plastic tubing connected to a blood bag, such as a sterile collection reservoir, for example a 250 mL collection bag, is then inserted into the placental vein. Alternatively, the tubing connected to the pump is inserted into the placenta vein, and the tube for the collection reservoir (s) is inserted into one or both of the placental arteries. The placenta is then perfused with a large amount of perfusion solution, for example, about 750 ml of perfusion solution. The cells in the perfusion fluid are then collected, for example, by centrifugation. In certain embodiments, perfusion solution, e.g., 100-300 mL perfusion solution, is perfused through the placenta to remove residual blood prior to perfusion to collect placental cells, such as placental stem cells and / or placental pluripotent cells. In another embodiment, the perfusion solution is not perfused in the placenta to remove residual blood prior to perfusion to collect placental cells.

In one embodiment, the proximal umbilical cord is held with forceps during perfusion, more preferably within 4-5 cm (centimeters) of the insertion of the cord into the placenta disc.

During the bleeding process, the primary collection of perfusion fluid from the mammalian placenta generally has a color of cord blood and / or placenta's residual red blood cells. As perfusion progresses and residual umbilical cord blood cells are washed from the placenta, the perfusion fluid becomes more colorless. In general, 30 to 100 ml (milliliters) of perfusion fluid is suitable for initially bleeding the placenta, but more or less perfusion fluid may be used depending on the observed results.

The volume of perfusion fluid used to isolate placental cells may vary depending on the number of cells collected, the size of the placenta, the number of collections made in a single placenta, and so on. In various embodiments, the volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, Lt; / RTI > mL. Typically, the placenta is perfused with 700-800 mL of perfusion fluid after bleeding.

The placenta can be perfused several times over several hours or days. If the placenta is to be perfused a number of times, it may be maintained or cultured in a container or other suitable container under sterile conditions and used in a cell collection composition or standard perfusion solution (e.g., an anticoagulant such as heparin, warfarin sodium, coumarin, (For example, 40-100 占 퐂 / ml), penicillin (for example,? -Mercaptoethanol (0.1 mM)) and / or antibiotics (E.g., phosphate buffered saline ("PBS")) with or without amphotericin B (e.g., 40 U / ml) . In one embodiment, the placenta is administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, The isolated placenta is maintained or cultured for a period of time without collecting the perfusion solution so that it is maintained or cultured for 19, 20, 21, 22, 23 or 24 hours, or 2 or 3 days or more. The perfused placenta can be used for more than one additional time (s), for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24 hours, or more, and then perfused for the second time with, for example, 700-800 mL of perfusion fluid. The placenta may be perfused at 1, 2, 3, 4, 5 or more times and may be perfused every 1, 2, 3, 4, 5 or 6 hours, for example. In a preferred embodiment, placental perfusion and collection of perfusion solution, e. G., Cell collection composition, is repeated until the number of recovered nucleated cells drops below 100 cells / ml. Different time points of perfusion fluid may be separately processed separately to recover a population of time-dependent cells, e. G., Stem cells. The perfusion fluids from different time points can also be collected. In a preferred embodiment, placental cells are collected once or several times between about 8 and about 18 hours after onset.

Preferably, it is not significantly more than the number obtained from the mammalian placenta that has not been perfused with the solution and has not been treated in another manner (e. G., Tissue destruction, e.g., enzymatic digestion) to obtain placental cells Placental cells are collected by perfusion. In this situation, "significantly more" means more than 10% more. For example, significantly more placental cells are produced by perfusion than the number of placental cells obtainable from the culture medium in which the placenta or a part thereof is cultured.

Placental cells can be isolated from the placenta by perfusion with a solution containing one or more proteases or other tissue-destructive enzymes. In a specific embodiment, the placenta or a portion thereof (e. G., Amniotic membrane, amniotic membrane and chorion, placental lobule or placental lobe, umbilical cord, or any combination of the foregoing) is made at 25-37 占 폚 and one or more tissue destructive enzymes ≪ / RTI > for 30 minutes in 200 mL of culture medium. Cells from the perfusate are collected, made to 4 ° C and washed with a mixture of cold inhibitors containing 5 mM EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. After a few minutes, the placental cells are washed with cold (e.g., 4 < 0 > C) stem cell collection composition.

5.3.5 Isolation, classification and characterization of placental cells

Regardless of whether it was obtained by perfusion or by physical destruction, for example by digestion with enzymes, the isolated placental cells, for example the cells described in Section 5.2.2 above, are first subjected to Ficoll concentration gradient centrifugation (I. E., Isolated) from other cells. Such centrifugation may follow any standard protocol for centrifugation rate and the like. In one embodiment, for example, cells collected from the placenta are recovered from the perfusion fluid by centrifugation at 5000 xg for 15 minutes at room temperature, which separates the cells, for example, from contaminating debris and platelets. In another embodiment, the placental perfusate is concentrated to about 200 ml, gently layered on the pillcol, and centrifuged at about 1100 xg for 20 minutes at 22 < 0 > C to collect the low density interface layer of the cells for further processing do.

The cell pellet is reconstituted in IMDM serum-free medium containing fresh stem cell collection composition, or a medium suitable for cell maintenance, such as stem cell maintenance, for example, 2 U / ml heparin and 2 mM EDTA (Gibco BRL, NY) It can be asked. For example, the total mononuclear cell fraction can be isolated using Lymphoprep (Nycomed Pharma, Oslo, Norway) according to the manufacturer's recommended procedure.

Placental cells obtained by perfusion or digestion can be isolated, for example, either separately or initially by differential trypsin treatment using 0.05% trypsin solution containing 0.2% EDTA (Sigma, St. Louis, Mo.) can do. Tissue culture Plastic-adherent isolated placenta cells are typically desorbed within about 5 minutes from the plastic surface, while differential trypsin treatment is possible because other adhesion populations typically require incubation in excess of 20-30 minutes . After using trypsin treatment and trypsin neutralization (e.g., using trypsin neutralization solution (TNS, Cambrex), etc.), desorbed placental cells can be harvested. In one embodiment to isolate adherent cells, an aliquot of, for example, about 5-10 x 10 ⁶ cells is loaded into each of several T-75 flasks, preferably T-75 flasks coated with fibronectin. In this embodiment, the cells can be cultured with commercially available mesenchymal stem cell growth medium (MSCGM) (Cambrex) and placed in a tissue culture incubator (37 ° C, 5% CO ₂ ). After 10-15 days, non-adherent cells are removed from the flask by washing with PBS. Subsequently, the PBS is replaced with MSCGM. Preferably, the flask is checked daily for the presence of various adherent cell types, particularly for identification and expansion of clusters of fibroblast-like cells.

The number and type of cells collected from the mammalian placenta can be determined, for example, by flow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue specific or cell marker specific antibodies), fluorescence activated cell sorting By examination of cytomorphology using optical microscopy or confocal microscopy, by measuring changes in morphology and cell surface markers using standard cell detection techniques such as fluorescence activated cell sorting (FACS), self-activating cell sorting (MACS), and And / or by measuring changes in gene expression using techniques known in the art, such as PCR and gene expression profiling. These techniques can also be used to identify positive cells for one or more specific markers. For example, using an antibody against CD34, it can be determined using the techniques described above whether the cell contains a detectable amount of CD34; If the cell contains a detectable amount of CD34, the cell is CD34 ⁺ . Similarly, if the cells produce enough OCT-4 RNA or OCT-4 RNA to be detectable by RT-PCR or significantly more than adult cells, the cell is OCT-4 ⁺ . Antibodies to cell surface markers (e.g. CD markers such as CD34) and sequences of stem cell-specific genes such as OCT-4 are known in the art.

Placental cells, particularly those isolated by phycolate separation, differential attachment, or a combination thereof, can be sorted using a fluorescence activated cell sorter (FACS). Fluorescence activated cell sorting (FACS) is a known method for isolating cell-containing particles, based on the fluorescent nature of the particles (Kamarch, 1987, Methods Enzymol, 151: 150-165). A small charge is generated by the laser excitation of the fluorescent moiety within the individual particles, causing the positive and negative particles to be electromagnetically separated from the mixture. In one embodiment, the cell surface marker-specific antibody or ligand is labeled with a different fluorescent label. The cells are processed through a cell sorter to separate the cells based on their ability to bind to the antibodies used. Particles classified as FACS can be deposited directly into separate wells of 96-well or 384-well plates to facilitate separation and cloning.

In one cell sorting scheme, cells from the placenta, for example, PDAC, are classified on the basis of the expression of one or more of the markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4 and / or HLA- do. This can be done in conjunction with procedures for selecting such cells based on the cell adhesion properties during incubation. For example, tissue culture plastic attachment selection can be made before or after classification based on marker expression. In one embodiment, for example, the cells are first sorted on the basis of expression of CD34; CD34 ^- cells have been maintained, further CD200 ^+, and HLA-G ^- a cell all other CD34 ^{- -} CD34 which are separated from the cells. In another embodiment, cells from the placenta are classified based on expression of the marker CD200 and / or HLA-G; For example, cells displaying CD200 and lacking HLA-G are isolated for further use. In a specific embodiment, for example, the expression of CD200 and / or the expression of an epitope in which, for example, HLA-G deficient cells express CD73 and / or CD105, or antibodies SH2, SH3 or SH4, RTI ID = 0.0 > CD38 < / RTI > or CD45. For example, In another embodiment, placental stem cells are CD200, HLA-G, CD73, CD105, CD34, and sorted by expression, or expression of the absence of CD38 and CD45, CD200 ^+, HLA-G ^-, CD73 ^+, CD105 ⁺ , CD34 ^- , CD38 ^-, and CD45 ^- are isolated from other placental cells for further use.

In a specific embodiment of any of the above embodiments of the classified placental cells, 50%, 60%, 70%, 80%, 90% or 95% or more of the cells in the remaining cell population are isolated placental cells . Placental cells can be sorted by one or more of the markers described in Section 5.2.2 above.

In a specific embodiment, for example, placental cells that are (1) adherent to tissue culture plastic and (2) placental cells that are CD10 ⁺ , CD34 ^- and CD105 ⁺ are sorted (ie isolated) from other placental cells. In another specific embodiment, (1) the tissue culture plastic and attached to the property, (2) CD10 ^+, CD34 ^-, CD105 ⁺ and CD200 ⁺ cells in the placenta is classified from other placental cells (i.e., are isolated). In another specific embodiment, (1) the tissue culture is attached to a plastic property, (2) CD10 ^+, CD34 ^-, CD45 ^-, CD90 ^+, CD105 ⁺ and CD200 ⁺ of placental cells are sorted from other placental cells (i. E. , ≪ / RTI >

For nucleotide sequence-based detection of placental cells, the sequences of the markers listed herein are readily available in public databases such as Gene Bank or EMBL.

Regarding antibody-mediated detection and classification of placental cells, e. G. Placental stem cells or placenta pluripotent cells, any antibody specific for a particular marker can be detected and sorted (e. G., Fluorescence-activated cell sorting) In combination with any suitable fluorophore or other label. Antibody / fluorophore combinations for specific markers include fluorescein isothiocyanate (FITC) conjugated monoclonal antibody to HLA-G (available from Serotec, Lalle, North Carolina); CD20 (available from BD Biosciences Pharmingen, San Jose, CA), and CD105 (available from BD Immunocytometry Systems, San Jose, Calif.), (Available from Minneapolis, Minn.); Picoerythrin (PE) conjugated monoclonal antibodies (BD Biosciences Farming) to CD44, CD200, CD117 and CD13; Picoerythrin-Cy7 (PE Cy7) conjugated monoclonal antibody (BD Biosciences Farming) for CD33 and CD10; Alopicoscien (APC) conjugated streptavidin and monoclonal antibody against CD38 (BD Biosciences Farming); And Bionylated CD90 (BD Biosciences Farming). Other antibodies that may be used include CD133-APC (Miltenyi), KDR-biotin (CD309, Abcam), cytokeratin K-Fitc (Sigma or Dako), HLA ABC-Fitc But are not limited to, HLA DR, DQ, DP-PE (BD), β-2-microglobulin-PE (BD), CD80-PE (BD) and CD86-APC (BD). Other antibody / label combinations that may be used include CD45-PerCP (peridineclorophyll protein); CD44-PE; CD19-PE; CD10-F (fluorescein); HLA-G-F and 7-amino-actinomycin-D (7-AAD); HLA-ABC-F, and the like. The list is not exhaustive and other antibodies from other sources are also commercially available.

The isolated placental cells provided herein can be assayed for CD117 or CD133 using, for example, picoeritrin-Cy5 (PE Cy5) conjugated streptavidin and biotin conjugated monoclonal antibodies against CD117 or CD133; Using this system, cells may appear to be positive for CD117 or CD133, respectively, due to their relatively high background.

Isolated placental cells may be labeled with antibodies to a single marker and detected and sorted. Placental cells can also be simultaneously labeled with different antibodies to different markers.

In another embodiment, the magnetic beads can be used to separate the cells. Cells can be sorted using a self-activating cell sorting (MACS), a method of sorting particles based on their ability to bind to magnetic beads (0.5-100 mu m in diameter). A variety of useful modifications can be performed on the magnetic microspheres, including adding covalently attached antibodies that specifically recognize specific cell surface molecules or haptens. The beads are then mixed with the cells to allow binding. The cells are then passed through a magnetic field to separate cells with specific cell surface markers. In one embodiment, such cells are then isolated and re-mixed with magnetic beads coupled to an antibody against an additional cell surface marker. Cells are passed back to the magnetic field to isolate cells bound to both antibodies. These cells can then be diluted into separate dishes, for example, microtiter plates for clonal isolation.

Isolated placental cells may also be characterized and / or sorted based on cytomorphology and growth characteristics. For example, isolated placental cells may be characterized and / or selected on the basis of this appearance, for example, to have a fibroblast-like appearance upon culture. The isolated placental cells may also be characterized and / or selected based on their ability to form embryo-analogs. For example, in one embodiment, placental cells that are in the form of fibroblasts and express CD73 and CD105, and which upon culture form one or more embryo-analogs, are isolated from other placental cells. In another embodiment, OCT-4 ⁺ placental cells that produce one or more embryo-analogs in culture are isolated from other placental cells.

In another embodiment, isolated placental cells can be identified and characterized by colony forming unit assays. Colony forming unit assays are commonly known in the art, such as MESENCULT ™ media (Stem Cell Technologies, Inc., Vancouver, BC, British Columbia).

Isolated placental cells can be obtained by standard techniques known in the art, for example, trypan blue exclusion assay, fluorescein diacetate absorption assay, propidium iodide absorption assay (for assessing viability); And thymidine uptake assays or MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) cell proliferation assays (to assess proliferation) Likelihood, and longevity. The lifespan can be determined by methods known in the art, for example, by determining the maximum value of population doubling during expansion.

The isolated placental cells, for example the isolated placental cells described in Section 5.2.2 above, may be used in conjunction with other techniques known in the art, such as selective growth of the desired cells (positive selection), selective destruction of unwanted cells ); Separation based on differential cellular cohesiveness within mixed populations, for example with soybean agglomerates; Frozen - thawing procedure; percolation; Conventional and zone centrifugation; Centrifugal purification (counter-streaming centrifugation); Unit weight separation; Countercurrent distribution; And can also be isolated from other placental cells using electrophoresis and the like.

5.4 Culture of Isolated Placental Cells

5.4.1 Culture medium

Cell cultures can be initiated or seeded using isolated placental cells, or a population of isolated placental cells, or cells or placental tissue from which placental cells have grown. In general, the cell can be an extracellular matrix or a ligand such as laminin, collagen (e.g. natural or modified), gelatin, fibronectin, ornithine, bitronectin, polylysine, CELLSTART ™, and / (Eg, Matrigel®, BD Discovery Labware, Bedford, Mass.), Or other suitable biomaterials or synthetic mimetic agents.

The isolated placental cells may be cultured in any of the media and under any conditions that are recognized in the art to be acceptable for the cultivation of cells, e. G., Stem cells. Preferably, the culture medium comprises serum. Isolated placental cells include, for example, ITS (insulin-transferrin-selenium), LA + BSA (linoleic acid-bovine serum albumin), dexamethasone L-ascorbic acid, PDGF, EGF, IGF-1, and penicillin / DMEM-LG (Dulbecco's modified essential medium, low-glucose) / MCDB 201 (chick fibroblast-based medium); DMEM-HG (high-glucose) containing 10% fetal bovine serum (FBS); DMEM-HG containing 15% FBS; IMDM (Iscove modified Dulbecco's medium) containing 10% FBS, 10% horse serum, and hydrocortisone; M199 comprising 1% to 20% FBS, EGF, and heparin; Alpha-MEM (minimal essential medium) containing 10% FBS, GLUTAMAX ™ and gentamicin; 10% FBS, Glutamax ™ and DMEM containing gentamicin, and the like.

Other media that can be used to culture placental cells include DMEM (high or low glucose), Eagle base medium, Ham F10 medium (F10), Ham F-12 medium (F12), Iscove transformed Dulbecco's medium, (MSCGM), Liebovitz L-15 medium, MCDB, DMEM / F12, RPMI 1640, improved DMEM (Gibco), DMEM / MCDB201 (Sigma), and cell- CELL-GRO FREE).

For example, serum (e.g., fetal bovine serum (FBS), preferably about 2-15% (v / v); horse serum (ES); human serum (HS)); Beta-mercaptoethanol (BME), preferably about 0.001% (v / v); (IGF-1), a leukemia inhibitory factor (IGF-1), or a combination of one or more growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor LIF), vascular endothelial growth factor (VEGF), and erythropoietin (EPO); Amino acids such as L-valine; And one or more antimicrobial agents and / or antifungal agents for controlling microbial contamination, such as penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either singly or in combination, Can be supplemented.

Isolated placental cells can be cultured in standard tissue culture conditions, for example, in tissue culture dishes or multiwell plates. Isolated placental cells can also be cultured using current methods. In this method, the isolated placental cells are suspended at about 1 x 10 ⁴ cells / mL in a medium of about 5 mL, and one or more medium droplets are placed in the tissue culture vessel, for example, inside the lid of a 100 mL Petri dish Is set. The droplet may be, for example, a single droplet or a number of droplets from, for example, a multi-channel pipettor. The lid is carefully inverted and placed on a plate bottom containing sterile PBS sufficient to maintain a large amount of liquid, for example, the moisture content in the dish atmosphere, and the stem cells are cultured.

In one embodiment, the isolated placental cells are cultured in the presence of a compound that acts to maintain the undifferentiated phenotype in the isolated placental cells. In a specific embodiment, the compound is substituted 3,4-dihydropyrimidol [4,5-d] pyrimidine. In another specific embodiment, the compound is a compound having the following chemical structure:

Such compounds may be contacted with isolated placental cells, or a population of isolated placental cells, for example, at a concentration of between about 1 [mu] M and about 10 [mu] M.

5.4.2 Expansion and proliferation of placental cells

Once isolated placental cells, or groups of isolated placental cells (e. G., Placental or placental cell populations isolated from more than 50% of placental cells normally associated with stem cells or stem cell populations in vivo) are obtained , Such a group of cells or cells can be proliferated and expanded in vitro. For example, a population of isolated placental cells may be cultured in tissue culture dishes, such as dishes, flasks, multiwell plates, etc., for a time sufficient for the cells to proliferate at a 70-90% Can be cultured to account for 70-90% of the cultured surface area of the tissue culture vessel.

The isolated placental cells can be seeded into the culture vessel at a density permitting cell growth. For example, cells can be seeded at a low density (e.g., from about 1,000 to about 5,000 cells / cm ² ) to a high density (e.g., at least about 50,000 cells / cm ² ). In a preferred embodiment, the cells are cultured in the presence of about 0 to about 5 vol% CO ₂ in air. In some preferred embodiments, the cells are cultured in about 2 to about 25% O ₂ in air, preferably about 5 to about 20% O ₂ in air. The cells are preferably cultured at about 25 캜 to about 40 캜, preferably at 37 캜. Preferably the cells are cultured in an incubator. The culture medium may be static, or may be stirred, for example, using a bioreactor. Preferably, placental cells such as placental stem cells or placental pluripotent cells are grown under low oxidative stress (e.g., with glutathione, ascorbic acid, catalase, tocopherol, N-acetylcysteine, etc.).

Once less than 100%, e. G., 70% to 90% total culturing rate is obtained, cells can be subcultured. For example, the cells can be treated with enzymes using techniques well known in the art, for example by trypsinization, to separate the cells from the tissue culture surface. After removing the cells by pipetting and counting the cells, about 10,000-100,000 cells / cm ² are subcultured into a fresh culture medium containing fresh culture medium. Typically, the new medium is of the same type as the medium from which the placenta cells isolated are removed. Isolated placental cells can be subcultured to approximately, at least, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20, .

5.4.3 Collection of Isolated Placental Cells

Also provided herein are isolated placental cells useful in the methods and compositions described herein, for example, a population of isolated placental cells described in Section 5.2.2 above. A population of isolated placental cells can be isolated directly from one or more placentas; That is, the cell population can be obtained from a perfusion fluid, or from a placental tissue contained within a perfusion fluid, or from a broken placental tissue, such as a placental tissue parasite (i.e., a collection of cells obtained by digestion with an enzyme of the placenta or a portion thereof) Or a group of placental cells comprising isolated placental cells contained within such packages. The isolated placental cells described herein may also be cultured and expanded to produce a population of isolated placental cells. A population of placental cells containing isolated placental cells may also be cultured and expanded to produce a placental cell population.

The placental cell population useful in the methods of treatment provided herein includes isolated placental cells, such as the isolated placental cells described in Section 5.4.2 herein. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of the cells in the placental cell population are isolated placental cells . That is, the population of isolated placental cells may be divided into a plurality of isolated placental cells, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% Cells that are not cells.

Isolated populations of placental cells useful in the methods and compositions described herein can be generated, for example, by selecting isolated placental cells derived from enzymatic digestion or perfusion, expressing specific markers and / or specific culture or morphological characteristics have. In one embodiment, for example, (a) attaching to a substrate, (b) selecting placental cells expressing CD200 and lacking expression of HLA-G; And isolating the cells from other cells to form a population of cells. In another embodiment, a population of cells is produced by selecting placental cells expressing CD200 and lacking expression of HLA-G, and isolating the cells from other cells to form cell populations. In another embodiment, a population of cells is produced by (a) attaching to a substrate, (b) selecting placental cells expressing CD73, CD105, and CD200, and isolating the cells from other cells to form cell populations . In another embodiment, a population of cells is produced by identifying placental cells expressing CD73, CD105 and CD200, and isolating the cells from other cells to form cell populations. In another embodiment, a method of producing a cell population comprises the steps of: (a) attaching to a substrate; (b) selecting placental cells expressing CD200 and OCT-4, isolating the cells from other cells to form a population of cells A cell population is produced. In another embodiment, a population of cells is produced by selecting placental cells expressing CD200 and OCT-4, and isolating the cells from other cells to form cell populations. (B) CD73 and CD105; and (c) the population of placental cells comprising stem cells is cultured under conditions permitting the formation of embryo-analogs. In another embodiment, A cell population is produced by selecting placental cells that promote the formation of one or more embryo-analogs in the population and isolating the cells from other cells to form a cell population. In another embodiment, CD73 and CD105 are expressed, and when a population of placental cells comprising stem cells is cultured under conditions permitting the formation of embryo-analogs, promoting the formation of one or more embryo-analogs in said population A placental cell is selected and a cell population is produced by isolating the cell from other cells to form a cell population. In another embodiment, there is provided a method of inhibiting the expression of HLA-G by (a) attaching to a substrate, (b) selecting placental cells expressing CD73 and CD105 and lacking expression of HLA-G, isolating the cells from other cells to form a population of cells A cell population is produced. In another embodiment, a population of cells is produced by selecting placental cells expressing CD73 and CD105 and lacking expression of HLA-G, and isolating the cells from other cells to form cell populations. In another embodiment, a method of producing a cell population comprises the steps of (a) adhering to a substrate, (b) expressing OCT-4, and (c) allowing a population of placental cells comprising stem cells to form an embryo-analog Selecting a placental cell that promotes the formation of one or more embryo-analogs in said population if cultured under conditions such that said placental cells are cultured under said conditions; And isolating the cells from other cells to form a population of cells. In another embodiment, when a population of placental cells that express OCT-4 and are cultured under conditions permitting the formation of embryo-analogs, the placenta that promotes the formation of one or more embryo-analogs in the population A cell population is produced by selecting a cell and isolating the cell from other cells to form a cell population.

In another embodiment, a cell population is produced by (a) attaching to a substrate, (b) selecting placental cells expressing CD10 and CD105 and not expressing CD34, isolating the cells from other cells to form a cell population Is produced. In another embodiment, a population of cells is produced by selecting placental cells expressing CD10 and CD105 and not expressing CD34, and isolating the cells from other cells to form cell populations. In another embodiment, the present invention provides a method of treating a cell, comprising: (a) attaching to a substrate; (b) selecting placental cells expressing CD10, CD105 and CD200 and not expressing CD34, isolating the cells from other cells, A group is produced. In another embodiment, a population of cells is produced by selecting placental cells expressing CD10, CD105 and CD200 and not expressing CD34, and isolating the cells from other cells to form cell populations. (B) selecting placental cells expressing CD10, CD90, CD105 and CD200 and not expressing CD34 and CD45, isolating the cells from other cells, Cell populations are produced by forming populations. In another specific embodiment, a population of cells is produced by selecting placental cells expressing CD10, CD90, CD105 and CD200 and not expressing CD34 and CD45, and isolating the cells from other cells to form cell populations.

Selected cell populations comprising placental cells with any of the marker combinations described in Section 5.2.2 above can be isolated or obtained in a similar manner.

In any of the above embodiments, the selection of an isolated cell population can be accomplished using ABC-p (placenta-specific ABC transporter protein; e.g., Allikmets et al., Cancer Res. 58 (23): 5337-9 )]) Of the placental cells may be further included. Such a method may also include, for example, selecting cells that exhibit one or more characteristics specific for mesenchymal stem cells, such as expression of CD44, expression of CD90, or a combination of the foregoing.

In such embodiments, the substrate may be any surface on which the cultivation and / or selection of cells, e. G. Isolated placental cells, can be achieved. Typically, the substrate is plastic, for example tissue culture dish or multiwell plate plastic. Tissue culture plastics can be coated with biomolecules, such as laminin or fibronectin.

Cells, e. G. Isolated placental cells, may be selected for placental cell populations by any means known in the art of cell selection. For example, cells can be selected using antibodies or antibodies to one or more cell surface markers, for example in flow cytometry or FACS. Selection can be achieved using antibodies with magnetic beads. Antibodies specific for particular stem cell-associated markers are known in the art. CD200 (Abcam), HLA-G (Abkam), CD73 (BD Biosciences Palmingen, San Diego, CA), CD105 (Abbeam, (BioDesign International, Sacho, Maine, USA). Antibodies against other markers are also commercially available, and antibodies to, for example, CD34, CD38 and CD45 are available, for example, from StemCell Technologies or Bio Design International.

The isolated placental cell population may include placental cells other than stem cells, or cells that are not placental cells.

The isolated cell population comprising the placenta-derived adherent cells described herein may comprise a second cell type, for example a placental cell other than a placenta-derived adherent cell, or a cell that is not, for example, a placental cell. For example, an isolated population of placenta-derived adherent cells can comprise a population of cells of a second type, for example, and can be combined therewith, wherein said cells of the second type are, for example, Cells, blood cells (e. G., Placental blood, placental blood cells, umbilical cord blood, umbilical cord blood cells, peripheral blood, peripheral blood cells, placental blood, cord blood from peripheral blood or the like) Cells (for example, stem cells isolated from placental blood, umbilical cord blood or peripheral blood), nucleated cells from placental perfusion fluid, for example, total nucleated cells from placental perfusion fluid; Derived stem cells, blood-derived nucleated cells, bone marrow-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, bone marrow-derived hematopoietic stem cells, crude bone marrow, adult (somatic) For example, a population of stem cells contained in cultured stem cells, a population of fully differentiated cells (for example, chondrocytes, fibroblasts, amniotic cells, osteoblasts, muscle cells, cardiac cells, etc.) . In a specific embodiment, the population of cells comprising placenta-derived adherent cells comprises placental stem cells or stem cells from the umbilical cord. In certain embodiments wherein the second type of cell is a blood or blood cell, the red blood cells have been removed from the cell population.

In a specific embodiment, the second type of cell is a hematopoietic stem cell. Such hematopoietic stem cells are, for example, in the unprocessed placenta, umbilical cord blood or peripheral blood; Within total nucleated cells from placental blood, umbilical cord blood or peripheral blood; Within the isolated population of CD34 ⁺ cells from placental blood, umbilical cord blood or peripheral blood; Within the unprocessed bone marrow; Within total nucleated cells from bone marrow; The isolated population of CD34 ⁺ cells from the bone marrow, and the like.

In another embodiment, an isolated population of placenta-derived adherent cells is combined with many adult or progenitor cells from the vascular system. In various embodiments, the cell is an endothelial cell, endothelial progenitor cell, myocardial cell, myocardial cell, pericyte cell, angiostatic cell, myoblast or myocardial cell.

In another embodiment, the second cell type is a non-embryonic cell type that is engineered in culture to express universal and functional markers associated with embryonic stem cells.

In a specific embodiment of said isolated population of placenta-derived adherent cells, one or both of the placenta-derived adherent cells and the second type of cells are autologous or allogeneic to the intended recipient of the cell.

In another specific embodiment, the composition comprises placenta-derived adherent cells and embryonic stem cells. In another specific embodiment, the composition comprises placenta-derived adherent cells and mesenchymal or stem cells, such as bone marrow-derived mesenchymal stem cells or stem cells. In another specific embodiment, the composition comprises bone marrow-derived hematopoietic stem cells. In another specific embodiment, the composition comprises placenta-derived adherent cells and hematopoietic progenitor cells, such as bone marrow, fetal blood, umbilical cord blood, placental blood, and / or peripheral blood cells from peripheral blood. In another specific embodiment, the composition comprises placenta-derived adherent cells and somatic stem cells. In a more specific embodiment, the somatic stem cells are neural stem cells, liver stem cells, pancreatic stem cells, endothelial stem cells, cardiac stem cells, or muscle stem cells.

In another specific embodiment, the second type of cell is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% . In another specific embodiment, the PDAC in the composition comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the cells in the composition. In another specific embodiment, the placenta-derived adherent cells comprise about, at least, or at most 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% of the cells in the population.

Cells in the isolated population of placenta-derived adherent cells can be divided into a number of different types of cells, for example, a population of stem cells, and a total number of nucleated cells within each population of about 100,000,000: 1, 50,000,000: 1, 20,000,000: 1, 10,000,000: 1, 5,000,000: 1, 2,000,000: 1, 1,000,000: 1, 500,000: 1, 200,000: 1, 100,000: 1, 50,000: 1, 20,000: 1000: 1, 500: 1, 200: 1, 100: 1, 50: 1, 20: 1, 10: 1, 5: 1, 2: 1, 1: 1; 1: 2; 1: 5; 1:10; 1: 100; 1: 200; 1: 500; 1: 1,000; 1: 2,000; 1: 5,000; 1: 10,000; 1: 20,000; 1: 50,000; 1: 100,000; 1: 500,000; 1: 1,000,000; 1: 2,000,000; 1: 5,000,000; 1: 10,000,000; 1: 20,000,000; 1: 50,000,000; Or in a ratio of about 1: 100,000,000. Cells within the isolated population of placenta-derived adherent cells can also be combined with many cells of many cell types.

In another embodiment, the placental cells described herein, e. G., The population of PDACs described above, are contacted with an osteogenic placenta adherent cell (OPAC), such as the method described in "Methods and Compositions for Treatment quot; amniotic membrane-derived angiogenic cells "in combination with OPAC as described in Patent Application No. 12 / 546,556 of " Bone Defects With Placental Stem Cells & No. 12 / 622,352, the disclosures of which are incorporated herein by reference in their entirety.

5.5 Production of Placental Cell Bank

The isolated cells from the postpartum placenta, for example the isolated placental cells described in section 5.2.2 above, can be cultured in a number of different ways to produce a lot set of isolated placental cells, A lot is a set of dosages that can be administered individually. Such a lot can be obtained, for example, from placental perfusate or from cells from the digested placental tissue with an enzyme. Lot sets of placental cells obtained from a number of placentas can be arranged in the bank of placental cells isolated, for example for long term storage. Generally, tissue culture plastic-adherent placental cells are obtained from an initial culture of placental material to form seed cultures, which expand under controlled conditions to form a population of approximately equal numbers of cells. The lot is preferably derived from a tissue of a single placenta, but can be derived from a tissue of multiple placentas.

In one embodiment, a placental cell lot is obtained as follows. First, placental tissue is broken down, for example, and digested with appropriate enzymes such as trypsin or collagenase (see section 5.3.3 above). Preferably the placental tissue comprises the entire amnion, the entire chorion, or both, for example from a single placenta, but may comprise only part of the amniotic membrane or chorion. The digested tissue is cultured for, for example, about 1 to 3 weeks, preferably about 2 weeks. After removing the non-adherent cells, the resulting dense colonies were collected by, for example, trypsinization. These cells are harvested, resuspended in a convenient volume of culture medium, and used for seeding into expanded cultures. The expanded culture may be any of the individual cell culture devices, for example, any arrangement of NUNC (TM) from Cell Factory. The cells are cultured in an expanded culture for example at a concentration of 1 x 10 ³ , 2 x 10 ³ , 3 x 10 ³ , 4 x 10 ³ , 5 x 10 ³ , 6 x 10 ³ , 7 x 10 ³ , 8 x 10 ³ , 9 ^{x 10 3, 1 x 10 4} , 1 x 10 4, 2 x 10 4, 3 x 10 4, 4 x 10 4, 5 x 10 4, 6 x 10 4, 7 x 10 4, 8 x 10 4, 9 x 10 ⁴ , or 10 x 10 ⁴ cells / cm ² are seeded. Preferably, about 1 x 10 ³ to about 1 x 10 ⁴ cells / cm ² are used to seed each expanded culture. The number of expanded cultures may be greater or lesser depending on the particular placenta (s) from which the cells are obtained.

The expanded culture is grown until the density of the cells in the culture reaches a certain value, for example about 1 x 10 ⁵ cells / cm ² . Cells can be harvested and cryopreserved at this point, or can be subcultured into new expanded cultures as described above. Cells can be subcultured prior to use, for example at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, have. Preferably a record of the cumulative number of population doublings is maintained during the expansion culture (s). Cells from the cultures were seeded at 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38 or 40 times, or 60 times. Preferably, however, the number of population doublings before dividing the population of cells into individual doses is from about 15 to about 30. The cells may be continuously cultured throughout the expansion process, or they may be frozen at one or more points during expansion.

Cells to be used for individual doses can be frozen, for example frozen, for future use. The individual doses may include, for example, from about 1 million to about 50 million cells / ml, and generally from about 10 ⁶ to about 10 ¹⁰ cells.

Thus, in one embodiment, expanding primary cultured placental cells from human postpartum placenta for a plurality of first population doublings; Cryopreserving the placenta cells to form a Master Cell Bank; Expanding a plurality of placental cells from a master cell bank for a plurality of second population doublings; Cryopreserving the placental cells to form a working cell bank; Expanding a plurality of placental stem cells from a working cell bank for a plurality of third population doubling; And cryopreserving the placental cells in individual doses, wherein the placental cell bank is produced by the method wherein the individual doses collectively constitute the placental cell bank. Optionally, a plurality of placental cells from said plurality of third group doubles can be expanded for a multiplicity of fourth population doublings and cryopreserved in individual doses, wherein said individual doses collectively constitute a placental cell bank do.

In another specific embodiment, said primary cultured placental cells comprise placental cells from placental perfusate. In another specific embodiment, said primary cultured placental cells comprise placental cells from digested placental tissue. In another specific embodiment, said primary cultured placental cells comprise placental perfusate and placental cells from digested placental tissue. In another specific embodiment, all of the placental cells in the placental cell primary culture are from the same placenta. In another specific embodiment, the method further comprises the step of selecting CD200 ⁺ or HLA-G ^- placental cells from said plurality of placental cells from said working cell bank to form individual doses. In another specific embodiment, said individual dose comprises about 10 < ⁴ > to about 10 ⁵ placental cells. In another specific embodiment, said individual dose comprises from about 10 ⁵ to about 10 ⁶ placental cells. In another specific embodiment, said individual dose comprises from about 10 ⁶ to about 10 ⁷ placental cells. In another specific embodiment, said individual dose comprises about 10 ⁷ to about 10 ⁸ placental cells. In another specific embodiment, the individual dose comprises about 10 ⁸ to about 10 ⁹ placental cells. In another specific embodiment, the individual capacity of from about 10 ⁹ to about 10 ¹⁰ placental cells.

In a preferred embodiment, the donor (e.g., parent) from which the placenta is obtained is tested for at least one pathogen. If the maternal is positive for the tested pathogen, discard the entire lot from the placenta. Such testing may be performed at any time during placental cell lot production, for example during an expansion culture. Pathogens tested for presence include hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, human immunodeficiency virus (type I and II), cytomegalovirus, herpes virus , But is not limited thereto.

5.6 Preservation of Placental Cells

The isolated placental cells, for example, the isolated placental cells described in Section 5.3.2 above, can be preserved, that is, under conditions that allow long-term storage, or under conditions that inhibit apoptosis or apoptotic cell death, for example .

Compositions comprising an apoptosis inhibitor, a necrosis inhibitor and / or an oxygen-carrying perfluorocarbon, for example as described in related US Application Publication No. 2007/0190042 (the disclosure of which is incorporated herein by reference in its entirety) Can be used to preserve placental cells. In one embodiment, a method of preserving a population of cells useful in the methods and compositions described herein comprises contacting the population of cells with a cell collection composition comprising an apoptosis inhibitor and an oxygen-carrying perfluorocarbon, wherein The apoptosis inhibitor is present in an amount sufficient to reduce or prevent apoptosis in the population of cells relative to the population of cells not contacted with the apoptosis inhibitor. In a specific embodiment, the apoptosis inhibitor is a caspase inhibitor. In another specific embodiment, the apoptosis inhibitor is a JNK inhibitor. In another specific embodiment, the JNK inhibitor does not modulate differentiation or proliferation of the cell. In another embodiment, the cell collection composition comprises the apoptosis inhibitor and the oxygen-carrying perfluorocarbon in separate phases. In another embodiment, the cell collection composition comprises the apoptosis inhibitor and the oxygen-carrying perfluorocarbon in an emulsion. In another embodiment, the cell collection composition further comprises an emulsifier, for example lecithin. In another embodiment, the apoptosis inhibitor and the perfluorocarbon are between about 0 ° C and about 25 ° C at the time of cell contact. In another specific embodiment, said apoptosis inhibitor and said perfluorocarbon are between about 2 ° C and 10 ° C or between about 2 ° C and about 5 ° C at the time of cell contact. In another specific embodiment, the contacting is performed during transport of the cell population. In another more specific embodiment, the contacting is performed during freezing and thawing of the population of cells.

A population of placental cells comprises, for example, contacting said population of cells with an apoptosis inhibitor and an organ-preservation compound, wherein said apoptosis inhibitor inhibits the expression of these cells in a population of cells that are not contacted with an apoptosis inhibitor In an amount sufficient to reduce or prevent apoptosis in the population, and for a time sufficient therefor. In a specific embodiment, the organ-preservation compound is a UW solution (also described in U.S. Patent No. 4,798,824; also known as ViaSpan), also see Southard et al., Transplantation 49 (2): 251-257 (1990)) or in U.S. Patent No. 5,552,267 (Stern et al.), The disclosures of which are incorporated herein by reference in their entirety. In another embodiment, the organ-preservation compound is hydroxyethyl starch, lactobionate, raffinos, or a combination thereof. In another embodiment, the cell collection composition further comprises an oxygen-carrying perfluorocarbon as a two-phase or as an emulsion.

In another embodiment of this method, the placental cells are contacted with a cell collection composition comprising an apoptosis inhibitor and oxygen-carrying perfluorocarbons, organ-preservation compounds or a combination thereof during perfusion. In another embodiment, the cells are contacted during a tissue destruction process, e.g., enzymatic digestion. In another embodiment, placental cells are contacted with the cell collection compound after collection by perfusion, or after collection by digestion with tissue disruption, e.g., enzymes.

Typically, during placental cell collection, enrichment and isolation, it is desirable to minimize or eliminate cellular stress due to hypoxia and mechanical stress. Thus, in another embodiment of this method, a population of cells or cells is exposed to hypoxic conditions during collection, enrichment or isolation for less than 6 hours during the storage, wherein the hypoxic condition is lower than normal blood oxygen concentration Concentration. In another specific embodiment, the population of cells is exposed to the hypoxic condition for less than 2 hours during the storage. In another specific embodiment, the population of cells is exposed to the hypoxic condition for less than 1 hour or less than 30 minutes, or is not exposed to hypoxic conditions during collection, concentration or isolation. In another specific embodiment, the population of cells is not exposed to shear stress during collection, fortification or isolation.

Placental cells can be cryopreserved and can be cryopreserved in, for example, cryopreservation media in small containers, such as ampoules. Suitable cryopreservation media include, but are not limited to, culture media including, for example, growth media, or cell freezing media, such as commercially available cell freezing media such as C2695, C2639 or C6039 (Sigma). The cryopreservation medium preferably contains DMSO (dimethylsulfoxide) at a concentration of, for example, from about 2% to about 15% (v / v), such as about 10% (v / v). The cryopreservation medium may comprise additional agonists such as methylcellulose and / or glycerol. Preferably the placental cells are cooled to about 1 [deg.] C / min during cryopreservation. The preferred cryopreservation temperature is from about -80 캜 to about -180 캜, preferably from about -125 캜 to about -140 캜. Cryopreserved cells can be transferred to liquid nitrogen prior to thawing for use. In some embodiments, for example when the ampoule reaches about -90 캜, they are transferred to a liquid nitrogen storage zone. Cryopreservation may also be achieved using a rate controlled freezer. The cryopreserved cells are thawed at a temperature of about 25 ° C to about 40 ° C, preferably about 37 ° C.

5.7 Composition comprising isolated placental cells

Herein, for example, the placental cells described in section 5.4.2 may be combined with any physiologically acceptable or medically acceptable compound, composition or device for use in the methods and compositions described herein. Compositions useful in the methods of treatment provided herein may include any one or more of the placental cells described herein (see Section 5.4.2 above). In certain embodiments, the composition is a pharmaceutical acceptable composition, for example, a composition comprising placental cells in a pharmaceutically acceptable carrier. See Section 5.9.2 below.

In certain embodiments, compositions comprising isolated placental cells additionally comprise a matrix, e. G., A cell removal matrix or a synthetic matrix. In another specific embodiment, the matrix is a three dimensional scaffold. In another specific embodiment, the matrix comprises collagen, gelatin, laminin, fibronectin, pectin, ornithine, or bitronectin. In another specific embodiment, the matrix is amniotic membrane or amniotic membrane-derived biomaterial. In another specific embodiment, the matrix comprises an extracellular membrane protein. In another specific embodiment, the matrix comprises a synthetic compound. In another specific embodiment, the matrix comprises a biologically active compound. In another specific embodiment, the biologically active compound is a growth factor, a cytokine, an antibody, or an organic molecule of less than 5,000 daltons.

In another embodiment, compositions useful in the therapeutic methods provided herein comprise a medium conditioned by any of the placental cells or by any of the placental cell populations.

5.7.1 Isolated cryopreserved placental cells

The isolated placental cell population useful in the methods and compositions described herein may be conserved, for example frozen, for further use. Methods for cryopreserving cells, such as stem cells, are known in the art. The isolated placental cell population may be prepared in a form that is readily administrable to an individual, for example, an isolated placental cell population is contained in a container suitable for medical use. Such a container can be, for example, a syringe, a sterile plastic bag, a flask, a bottle or other container from which the isolated placental cell population can be easily dispensed. For example, the container may be a blood bag, or other medically acceptable plastic bag suitable for intravenous administration of liquid to a recipient. In certain embodiments, the container enables cryopreservation of the combined cell population.

The cryopreserved isolated placental cell population may comprise isolated placental cells derived from a single donor or from a plurality of donors. The isolated placental cell population can be fully matched for HLA or intended to partially or completely mismatch HLA for the intended recipient.

Thus, in one embodiment, the isolated placental cells may be used in the methods described herein in the form of a composition comprising a tissue culture platelet-adherent placental cell population in a vessel. In a specific embodiment, the isolated placental cells are cryopreserved. In another specific embodiment, the container is a bag, flask, or bottle. In another specific embodiment, the bag is a sterile plastic bag. In another specific embodiment, the bag is suitable, permits or facilitates intravenous administration of the isolated placental cell population, for example, by intravenous infusion. The bag may comprise multiple lumens or compartments interconnected to allow for the mixing of placental cells isolated prior to or during administration with one or more other solutions, e. G., A drug. In another specific embodiment, the composition comprises one or more compounds that facilitate cryopreservation of the combined cell population. In another specific embodiment, said isolated placental cell population is contained in a physiologically acceptable aqueous solution. In another specific embodiment, the physiologically acceptable aqueous solution is a 0.9% NaCl solution. In another specific embodiment, said isolated placental cell population comprises placental cells that are HLA matched to the recipient of said cell population. In another specific embodiment, the combined cell population comprises placental cells that are at least partially HLA mismatched to the population of the cell population. In another specific embodiment, the isolated placental cells are derived from a plurality of donors.

In certain embodiments, the isolated placental cells in the container are isolated CD10 ⁺ , CD34 ^- , CD105 ⁺ placental cells that have been cryopreserved and contained in a container. In a specific embodiment, the CD10 ⁺ , CD34 ^- , CD105 ⁺ placental cells are also CD200 ⁺ . In another specific embodiment, the CD10 ⁺ , CD34 ^- , CD105 ⁺ , CD200 ⁺ placental cells are also CD45 ^- or CD90 ⁺ . In another specific embodiment, the CD10 ⁺ , CD34 ^- , CD105 ⁺ , CD200 ⁺ placental cells are also CD45 ^- or CD90 ⁺ . In another specific ^{embodiment, CD34 -, CD10 +, CD105} + placental cells are added to the ^{CD13 +, CD29 +, CD33 +} , CD38 -, CD44 +, CD45 -, CD54 +, CD62E -, CD62L -, CD62P -, ^{SH3 + (CD73 +), SH4} + (CD73 +), CD80 -, CD86 -, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD117 -, CD144 / VE- cadherin ^about, CD184 / CXCR4 ^{- , CD200 +, CD133 -, OCT} -4 +, SSEA3 -, SSEA4 -, ABC-p +, KDR - (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ, DR -, HLA- G ^- is or programmed death ligand -1 (PDL1) ⁺ one or more, or any combination thereof of the. In another specific ^{embodiment, CD34 -, CD10 +, CD105} + placental cells are added to the ^{CD13 +, CD29 +, CD33 +} , CD38 -, CD44 +, CD45 -, CD54 / ICAM +, CD62E -, CD62L -, CD62P ^{-, SH3 + (CD73 +)} , SH4 + (CD73 +), CD80 -, CD86 -, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD117 -, CD144 / VE- cadherin ^about, CD184 / ^{CXCR4 -, CD200 +, CD133 -} , OCT-4 +, SSEA3 -, SSEA4 -, ABC-p +, KDR - (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ, DR -, HLA-G < ^- & gt ^; and the programmed death-1 ligand (PDL1) ⁺ .

In another specific embodiment, the above-mentioned isolated placental cells are isolated CD200 ⁺ , HLA-G ^- placental cells that have been cryopreserved and contained in a container. In another embodiment, the isolated placental cells are CD73 ⁺ , CD105 ⁺ , CD200 ⁺ cells that have been cryopreserved and contained in a container. In another embodiment, the isolated placental cells are CD200 ⁺ , OCT-4 ⁺ stem cells that have been cryopreserved and contained in a container. In another embodiment, the isolated placental cells are cryopreserved and CD73 ⁺ , CD105 ⁺ cells contained in a container, wherein said isolated placental cells are cultured with a population of placental cells under conditions permitting the formation of embryo-analogs Lt; RTI ID = 0.0 > embryo-like < / RTI > The ^cells In yet another embodiment, the isolated placental cells, the ^{CD73 +, CD105 +, HLA-} G contained in the container was frozen and stored. In another embodiment, the isolated placental cells are cryopreserved and are OCT-4 ⁺ placental cells contained in a container, wherein the cells are incubated with a population of placental cells under conditions permitting the formation of embryo-analogs, Promoting the formation of the above embryo-analog.

In another specific embodiment, the isolated placental cells are CD34 mentioned above, when detected by a flow ^cytometry-a, CD10 ⁺ and CD105 ⁺ stem cells of the placenta or placental pluripotent cells (e.g., PDAC). In another specific embodiment, the isolated CD34 ^-, CD10 ^+, CD105 ⁺ placental cells have the potential to differentiate into cells with cells, or cartilage forming cells having a phenotype, osteogenic phenotype having a neural phenotype. In another specific embodiment, the isolated CD34 ^-, CD10 ^+, CD105 ⁺ placental cells are CD200 ⁺ additionally. In another specific embodiment, the isolated CD34 ^- , CD10 ⁺ , CD105 ⁺ placental cells are CD90 ⁺ or CD45 ^- when further detected by flow cytometry. In another specific embodiment, the isolated CD34 ^- , CD10 ⁺ , CD105 ⁺ placental cells are CD90 ⁺ or CD45 ^- when further detected by flow cytometry. In another specific embodiment, the CD34 ^- , CD10 ⁺ , CD105 ⁺ , CD200 ⁺ placental cells are CD90 ⁺ or CD45 ^- when further detected by flow cytometry. In another specific embodiment, CD34 ^- , CD10 ⁺ , CD105 ⁺ , CD200 ⁺ cells are CD90 ⁺ and CD45 ^- when further detected by flow cytometry. In another specific ^{embodiment, CD34 -, CD10 +, CD105} +, CD200 +, CD90 +, CD45 - cells when added to the detection by flow cytometry into CD80 ^- and CD86 ^- a. In another specific embodiment, CD34 ^-, CD10 ^+, CD105 ⁺ cells are added to the CD29 ^+, CD38 ^- is at least one of ⁺ SH3 or ^{SH4 + -, CD44 +, CD54} +, CD80 -, CD86. In another specific embodiment, the cell is further CD44 ⁺ . Wherein any of the isolated CD34 ^-, from CD10 ^+, specific embodiments of CD105 ⁺ placental cells, the cells are added to the ^{CD117 -, CD133 -, KDR -} (VEGFR2 -), HLA-A, B, C +, HLA-DP , DQ, DR ^- and / or PDL1 ⁺ .

In a specific embodiment of any of the above cryopreserved isolated placental cells, the container is a bag. In various embodiments, the container comprises at least or at most 1 x 10 ⁶ of the isolated placental cells, 5 x 10 ⁶ of the isolated placental cells, 1 x 10 ⁷ of the isolated placental cells, 5 x 10 ⁷ wherein the isolated placental cells, 1 x 10 ⁸ of said the isolated placental cells, 5 x 10 ⁸ of said the isolated placental cells, 1 x 10 ⁹ of said isolated placental cells, 5 x 10 ⁹ of the an isolated placenta Cells, 1 x 10 ¹⁰ said isolated placental cells, or 1 x 10 ¹⁰ said isolated placental cells. In another specific embodiment of any of the above cryopreserved populations, the isolated placental cells were subcultured approximately, at least, or at most 5 times, or at most 10 times, at most 15 times, or at most 20 times. In another specific embodiment of any of the above cryopreserved isolated placental cells, the isolated placental cells have been expanded in the container.

In a specific embodiment, the single unit dose of placenta-derived adherent cells is about, at least, or at most 1 x 10 ⁵ , 5 x 10 ⁵ , 1 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , ^{x 10 7, 1 x 10 8} , 5 x 10 8, 1 x 10 9, 5 x 10 9, 1 x 10 10, 5 x 10 10, 1 x 10 11 , or may include a placenta-derived adherent cells of the excess have. In certain embodiments, the pharmaceutical compositions provided herein comprise a population of placenta-derived adherent cells comprising 50% or more viable cells (i.e., at least 50% of the cells in the population are functional or alive). Preferably, more than 60% of the cells in the population are viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the cells in the population in the pharmaceutical composition are viable.

5.7.2 Genetically engineered placental cells

Also provided herein is a pharmaceutical composition comprising a placental cell, such as any placental pluripotent cell, or a placental cell according to section 5.2.2, or such a placental cell, wherein said placental cell is associated with angiogenesis, or Are genetically engineered to produce recombinant or exogenous cytokines that promote it. In certain embodiments, the protein that promotes angiogenesis is selected from the group consisting of HGF, VEGF (e.g., VEGFD), fibroblast growth factor (FGF) (ANG), epidermal growth factor (EGF), epithelial-neutrophil-activating protein 78 (ENA-78), polystatin, granulocyte colony-stimulating factor (G- , Interleukin-6 (IL-6), IL-8, leptin, monocyte chemoattractant protein-1 (MCP-1), MCP-3, platelet-derived growth factor subunit B (PDGFB) Is one or more of the following: Tumor Beta 1 (TGF-? 1), Trombopoietin (Tpo), Metoloproteinase 1 (TIMP1), Tissue Inhibitor of TIMP2 and / or Urokinase Plasminogen Activator Receptor .

For example, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, genetically manipulating cells using polyethylene glycol, or other methods known to those skilled in the art can be used. These include expression vectors that transport and express nucleic acid molecules into cells. (Geoddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transformation or transfection of host cells are described in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)], and other experimental textbooks.

The placental cells, for example the PDACs described in section 5.2 above, can be used for the treatment of, for example, DNA or RNA viral vectors such as retroviruses (including lentiviruses), monkey viruses 40 (SV40), adenoviruses, ≪ / RTI > virus transfer; Chemical delivery, such as calcium phosphate transfection and DEAE dextran transfection methods; Membrane fusion delivery using, for example, DNA-loading membrane vesicles, such as liposomes, erythrocyte ghosts, and protoplasts; Or by introducing DNA or RNA, e. G. DNA or RNA encoding the protein of interest, into the cell by a method including physical delivery techniques such as microinjection, electroporation, or naked DNA delivery . Placental cells can be genetically altered by insertion of exogenous DNA, or by replacement of a section of the cell genome with exogenous DNA. Insertion of the exogenous DNA sequence (s) can be achieved, for example, by homologous recombination or by viral integration into the host cell genome, or into the cells of DNA using plasmid expression vectors and nuclear localization sequences, Can be performed by integration. DNA may include one or more promoters that allow positive or negative induction of expression of a protein of interest using a particular chemical / drug, e.g., tetracycline; In another embodiment, the promoter may be constitutive.

Calcium phosphate transfection can be used, for example, to introduce plasmid DNA containing polynucleotide sequences encoding proteins of interest into cells. In certain embodiments, the DNA is combined with a solution of calcium chloride and then added to the phosphate-buffered solution. Once the precipitate forms, the solution is added directly to the cultured cells. Treatment with DMSO or glycerol can be used to improve transfection efficiency and the level of stable transfectants can be improved using bis-hydroxyethylamino ethanesulfonate (BES). Calcium phosphate transfection systems are commercially available (for example, PROFECTION®, Promega Corp., Madison, Wis.). DEAE-dextran transfection can also be used.

Isolated placental cells can also be manipulated genetically by microinjection. In certain embodiments, the glass micropipette is guided into the nucleus of the cell under an optical microscope to inject DNA or RNA.

In addition, placental cells can be genetically modified using electroporation. In certain embodiments, DNA or RNA is added to a suspension of cultured cells, a DNA / RNA-cell suspension is placed between the two electrodes and an electric pulse is applied to make the outer membrane of the cell temporarily permeable, It is evident by the occurrence of the pore crossing.

Liposomal delivery of DNA or RNA to genetically modify cells can optionally be accomplished by the use of dioloylphosphatidylethanolamine (DOPE) or dioloylphosphatidylcholine (DOPC), such as LIPOFECTIN (Life Technologies, Inc., Life Technologies, Inc.). ≪ / RTI > Other commercially available delivery systems include, but are not limited to, EFFECTENE ™ (Qiagen), DOTAP (Roche Molecular Biochemicals), FUGENE 6 ™ , And TRANSFECTAM (Promega). ≪ / RTI >

Viral vectors can be used to genetically alter placental cells, for example, by delivery of a target gene, polynucleotide, antisense molecule or ribozyme sequence into a cell. Although retroviral vectors are effective in transducing rapidly dividing cells, many retroviral vectors have also been developed to effectively deliver DNA into non-dividing cells. Packaging cell lines for retroviral vectors are known to those skilled in the art. In certain embodiments, the retroviral DNA vector has a first LTR at 5 'of the SV40 promoter operably linked to the target gene sequence cloned into the multiple cloning site, followed by two retroviruses such that the second LTR is located at 3' Lt; / RTI > Once formed, the retroviral DNA vector is transferred into the packaging cell line using calcium phosphate-mediated transfection as previously described. After about 48 hours of virus production, virus vectors containing target gene sequences were now collected. Methods for transfecting cells using lentiviral vectors, recombinant herpesviruses, adenoviral vectors, or alphavirus vectors are known in the art.

Successful transfection or transfection of target cells can be demonstrated using genetic markers as techniques known to those skilled in the art. For example, the green fluorescent protein of Aequorea victoria has been shown to be an effective marker for identifying and tracking genetically modified hematopoietic cells. Other selectable markers include a beta-Gal gene, a truncated nerve growth factor receptor, or a drug selection marker (including, but not limited to, NEO, MTX, or hygromycin).

5.7.3 Pharmaceutical composition

A population of isolated placental cells, for example a population of PDACs, or cells comprising isolated placental cells, may be formulated in vivo, for example, as a pharmaceutical composition for use in the methods of treatment provided herein. Such a pharmaceutical composition may comprise a population of placental cells isolated in a pharmaceutically acceptable carrier, for example a saline solution or another acceptable physiologically acceptable solution for in vivo administration, or a population of cells comprising isolated placental cells . The pharmaceutical composition comprising the isolated placental cells described herein may comprise any or any combination of isolated placental cell populations or isolated placental cells described elsewhere herein. The pharmaceutical composition may comprise an isolated placental cell of the fetus, an isolated placental cell of the mother, or an isolated placental cell of both the fetus and the host. The pharmaceutical compositions provided herein may further comprise isolated placental cells obtained from a single individual or placenta or from a plurality of individuals or placenta.

The pharmaceutical compositions provided herein may comprise any number of isolated placental cells. In various embodiments, for example, the single unit dose of isolated placental cells may be at least about 1 x 10 ⁵ , 5 x 10 ⁵ , 1 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ , 5 x 10 ⁸ , 1 x 10 ⁹ , 5 x 10 ⁹ , 1 x 10 ¹⁰ , 5 x 10 ¹⁰ , 1 x 10 ¹¹ or more isolated placental cells .

The pharmaceutical compositions provided herein comprise a population of cells comprising 50% viable cells or more (i. E., At least 50% of the population are functioning or alive). Preferably, more than 60% of the cells in the population are viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the cells in the population in the pharmaceutical composition are viable.

The pharmaceutical compositions provided herein include, for example, one or more compounds (e. G., Anti-T-cell receptor antibodies, immunosuppressants, etc.) that facilitate engraftment; Stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, plasma light, and the like.

When formulated as a injectable solution, in one embodiment, the pharmaceutical composition comprises about 1% to 1.5% HSA and about 2.5% dextran. In a preferred embodiment, the pharmaceutical composition is 5% HSA and containing 10% dextran and (e.g., 10 mg / kg) immunosuppressive agents, e.g., cyclosporin A from about 5 x 10 ⁶ in the solution containing optionally one Cells / milliliter to about 2 x 10 ⁷ cells / milliliter.

In another embodiment, a pharmaceutical composition, e. G., A solution, comprises a plurality of cells, e. G., Isolated placental cells, e. G., Placental stem cells or placenta pluripotent cells, 10 ⁶ cells / milliliter to about 5.0 +/- 1.5 x 10 ⁶ cells / milliliter. In another embodiment, the pharmaceutical composition comprises about 1.5 x 10 ⁶ cells / milliliter to about 3.75 x 10 ⁶ cells / milliliter. In another embodiment, the pharmaceutical composition comprises about 1 x 10 ⁶ cells / mL to about 50 x 10 ⁶ cells / mL, about 1 x 10 ⁶ cells / mL to about 40 x 10 ⁶ cells / mL, about 1 about 10 x ⁶ cells / mL to about 30 x 10 ⁶ cells / mL, about 1 x 10 ⁶ cells / mL to about 20 x 10 ⁶ cells / mL, about 1 x 10 ⁶ cells / mL to about 15 x 10 ⁶ cells / mL, or about 1 x 10 ⁶ cells / mL to about 10 x 10 ⁶ cells / mL. In certain embodiments, the pharmaceutical composition does not comprise a visible cell mass (i.e., no large cell mass), or substantially no such visible mass. As used herein, "large cell clumps" refers to agglomerates of cells that are visible, e.g., macroscopically visible, without enlargement, and generally refers to cell aggregates larger than about 150 micrometers. In some embodiments, the pharmaceutical composition comprises about 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5% , 9.5% or 10% dextran, such as dextran-40. In a specific embodiment, the composition comprises about 7.5% to about 9% dextran-40. In a specific embodiment, the composition comprises about 5.5% dextran-40. In certain embodiments, the pharmaceutical composition comprises from about 1% to about 15% human serum albumin (HSA). In a specific embodiment, the pharmaceutical composition comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% Or 15% HSA. In a specific embodiment, the cells were cryopreserved and thawed. In another specific embodiment, the cells were filtered through 70 [mu] m to 100 [mu] m filters. In another specific embodiment, the composition does not comprise a visible cell mass. In another specific embodiment, the composition comprises less than about 200 cell clumps per 10 ⁶ cells, wherein the cell clumps are visible only under a microscope, e.g., under an optical microscope. In another specific embodiment, the composition comprises less than about 150 cell clusters per 10 ⁶ cells, wherein the cell clumps are visible only under a microscope, e.g., under an optical microscope. In another specific embodiment, the composition comprises less than about 100 cell clusters per 10 ⁶ cells, wherein the cell clumps are visible only under a microscope, e.g., under an optical microscope.

In a specific embodiment, the pharmaceutical composition comprises about 1.0 ± 0.3 × 10 ⁶ cells / milliliter, about 5.5% dextran-40 (w / v), about 10% HSA (w / v), and about 5% DMSO / v).

In another embodiment, the pharmaceutical composition comprises a plurality of cells, for example, contain a plurality of the isolated placental cells in a solution containing 10% dextran -40, at which time the pharmaceutical composition comprises from about 1.0 ± 0.3 x 10 ⁶ cells / Milliliter to about 5.0 +/- 1.5 x 10 ⁶ cells / milliliter, and the composition does not include a macroscopically visible cell mass (i.e., does not contain large cell masses). In some embodiments, the pharmaceutical composition comprises about 1.5 x 10 ⁶ cells / milliliter to about 3.75 x 10 ⁶ cells / milliliter. In a specific embodiment, the cells were cryopreserved and thawed. In another specific embodiment, the cells were filtered through 70 [mu] m to 100 [mu] m filters. In another specific embodiment, the composition comprises less than about 200 microcellular masses per 10 ⁶ cells (i. E., A cell mass only visible when enlarged). In another specific embodiment, the pharmaceutical composition comprises less than about 150 microcellular lumps per 10 ⁶ cells. In another specific embodiment, the pharmaceutical composition comprises less than about 100 microcellular lumps per 10 ⁶ cells. In another specific embodiment, the pharmaceutical composition comprises a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition comprises 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% DMSO or less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% DMSO.

Compositions comprising cells, which are produced by one of the methods disclosed herein, are further provided herein. For example, in one embodiment, the pharmaceutical composition comprises cells wherein the solution comprising placental cells, such as placental stem cells or placental pluripotent cells, is filtered to form a filtered cell-containing solution; Prior to cryopreservation, for example, the filtered cell-containing solution may be stored at a volume of about 1 to 50 x 10 ⁶ , 1 to 40 x 10 ⁶ , 1 to 30 x 10 ⁶ , 1 to 20 x 10 ⁶ , 1 to 15 x 10 ⁶ , or 1 to 10 x 10 ⁶ cells with the first solution; And diluting the resulting filtered cell-containing solution with a second solution containing dextran but not human serum albumin (HSA) to produce the pharmaceutical composition. In certain embodiments, it diluted with up to about 15 x 10 ⁶ cells / ml. In certain embodiments, it has been diluted to about 10 3 x 10 ⁶ cells / milliliter or less. In certain embodiments, and it diluted to about 7.5 x 10 ⁶ cells / ml. In another specific embodiment, the filtered cell-containing solution prior to dilution in the case of including around 15 x 10 less than ⁶ cells / ml in a filter is arbitrary. In another particular embodiment, the filtration is optional if the filtered cell-containing solution before dilution comprises less than about 10 3 x 10 ⁶ cells / milliliter. In another particular embodiment, the filtration is optional if the filtered cell-containing solution comprises less than about 7.5 x 10 ⁶ cells / milliliter prior to dilution.

In a specific embodiment, the cells are cryopreserved between the dilution by the first dilution solution and the dilution by the second dilution solution. In another specific embodiment, the first dilute solution comprises dextran and HSA. The dextran in the first diluting solution or the second diluting solution may be dextran of any molecular weight, for example, dextran having a molecular weight of about 10 kDa to about 150 kDa. In some embodiments, the dextran in the first diluent solution or the second solution is about 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0% , 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or 10% dextran. In another specific embodiment, the dextran in the first dilution solution or the second dilution solution is dextran-40. In another specific embodiment, the dextran in the first dilution solution and the second dilution solution is dextran-40. In another specific embodiment, the dextran-40 in the first dilute solution is 5.0% dextran-40. In another specific embodiment, the dextran-40 in the first dilute solution is 5.5% dextran-40. In another specific embodiment, the dextran-40 in the second dilute solution is 10% dextran-40. In another specific embodiment, the HSA in the solution comprising HSA is 1-15% HSA. In a specific embodiment, the HSA in the solution comprising HSA comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 12%, 13%, 14% or 15% HSA. In another specific embodiment, the HSA in the solution comprising HSA is 10% HSA. In another specific embodiment, the first dilute solution comprises HSA. In another specific embodiment, the HSA in the first diluent solution is 10% HSA. In another specific embodiment, the first diluent solution comprises a cryoprotectant. In another specific embodiment, the cryoprotectant is DMSO. In another specific embodiment, the dextran-40 in the second dilute solution is about 10% dextran-40. In another specific embodiment, the composition comprising cells comprises about 7.5% to about 9% dextran. In another specific embodiment, the pharmaceutical composition comprises about 1.0 + - 0.3 x 10 ⁶ cells / milliliter to about 5.0 + 1.5 x 10 ⁶ cells / milliliter. In another specific embodiment, the pharmaceutical composition comprises about 1.5 x 10 ⁶ cells / milliliter to about 3.75 x 10 ⁶ cells / milliliter.

In another embodiment, there is provided a method of producing a cell-containing solution, comprising: (a) filtering a cell-containing solution comprising placental cells, such as placental stem cells or placental pluripotent cells, before cryopreservation to produce a filtered cell- (b) contacting the cells in the filtered cell-containing solution with about 1 to 50 x 10 ⁶ , 1 to 40 x 10 ⁶ , 1 to 30 x 10 ⁶ , 1 to 20 x 10 ⁶ , 1 to 15 x 10, ⁶ , or 1 to 10 x 10 ⁶ cells / milliliter; (c) thawing the cells; And (d) diluting the filtered cell-containing solution with a dextran-40 solution at about 1: 1 to about 1: 11 (v / v). In certain embodiments, if the number of cells is less than about 10 3 x 10 ⁶ cells / milliliter prior to step (a), filtration is optional. In another specific embodiment, the cells of step (b) are cryopreserved at about 10 3 x 10 ⁶ cells / ml. In another specific embodiment, the cells of step (b) are cryopreserved in a solution comprising about 5% to about 10% dextran-40 and HSA. In certain embodiments, diluted in step (b) to about 15 x 10 ⁶ cells / ml.

(A) suspending placental cells, for example placental stem cells or placenta pluripotent cells, in a 5.5% dextran-40 solution containing 10% HSA to form a cell-containing solution; (b) filtering the cell-containing solution with a 70 占 퐉 filter; (c) contacting the cell-containing solution with a solution comprising 5.5% dextran-40, 10% HSA and 5% DMSO at a concentration of about 1 to 50 x 10 ⁶ , 1 to 40 x 10 ⁶ , 1 to 30 x 10 ⁶ 1 to 20 x 10 ⁶ , 1 to 15 x 10 ⁶ , or 1 to 10 x 10 ⁶ cells per milliliter; (d) cryopreserving the cells; (e) thawing the cells; And (f) diluting the cell-containing solution with 10% dextran-40 in a ratio of 1: 1 to 1:11 (v / v). Diluted in. In certain embodiments, step (c) is about 15 x 10 ⁶ cells / ml or less. In certain embodiments, the dilution in step (c) is less than or equal to about 10 3 x 10 ⁶ cells / mL. Diluted in. In certain embodiments, step (c) is about 7.5 x 10 ⁶ cells / mL or less.

In another embodiment, a composition comprising cells comprises: (a) centrifuging a plurality of cells to collect cells; (b) resuspending the cells in 5.5% dextran-40; (c) centrifuging the cells to collect cells; (d) resuspending the cells in a 5.5% dextran-40 solution containing 10% HSA; (e) filtering the cells with a 70 占 퐉 filter; (f) the cells are cultured at about 1 to 50 x 10 ⁶ , 1 to 40 x 10 ⁶ , 1 to 30 x 10 ⁶ , 1 to 20 x 10 ⁶ in 5.5% dextran-40, 10% HSA and 5% ⁶ , 1 to 15 x 10 ⁶ , or 1 to 10 x 10 ⁶ cells per milliliter; (g) cryopreserving the cells; (h) thawing the cells; And (i) diluting the cells with 1: 1 to 1: 11 (v / v) with 10% dextran-40. In certain embodiments, diluted in step (f) up to about 15 x 10 ⁶ cells / ml. In certain embodiments, step (f) is diluted to less than about 10 3 x 10 ⁶ cells / mL. In certain embodiments, the dilution in step (f) is less than or equal to about 7.5 x ¹⁰⁶ cells / mL. In another particular embodiment, if the number of cells is less than about 10 3 x 10 ⁶ cells / milliliter, filtration is optional.

Compositions comprising the isolated placental cells described herein, e. G., Pharmaceutical compositions, may comprise any of the isolated placental cells described herein.

Other injectable formulations suitable for administration of cellular products may be used.

In one embodiment, the pharmaceutical composition comprises isolated placental cells that are substantially or completely of non-maternal origin, i. E. Having a fetal genotype, for example at least about 90%, 95%, 98%, 99% % Is non-mature origin. For example, in one embodiment, the pharmaceutical compositions are CD200 ^+, and HLA-G ^-; CD73 ⁺ , CD105 ⁺ , and CD200 ⁺ ; CD200 ⁺ and OCT-4 ⁺ ; CD73 ^+, CD105 ^+, and HLA-G ^-; CD73 ⁺ and CD105 ⁺ and promoting the formation of one or more embryo-analogs in a population of placental cells comprising a population of the isolated placental cells when the population of placental cells is cultured under conditions permitting the formation of embryo-analogs A group of isolated placental cells; Or OCT-4 ⁺ , promoting the formation of one or more embryo-analogs in a population of placental cells comprising a population of the isolated placental cells when the population of placental cells is cultured under conditions permitting the formation of embryo-analogs A group of isolated placental cells; Or a combination thereof, wherein at least 70%, 80%, 90%, 95% or 99% of said isolated placental cells are non-maternal origin. In another embodiment, the pharmaceutical composition comprises CD10 ⁺ , CD105 ⁺ and CD34 ^- ; CD10 ⁺ , CD105 ⁺ , CD200 ⁺ and CD34 ^- ; CD10 ⁺ , CD105 ⁺ , CD200 ⁺ , CD34 ^- , CD90 ⁺ or CD45 ^- ; CD10 ⁺ , CD90 ⁺ , CD105 ⁺ , CD200 ⁺ , CD34 ^- and CD45 ^- ; CD10 ⁺ , CD90 ⁺ , CD105 ⁺ , CD200 ⁺ , CD34 ^- and CD45 ^- ; CD200 ^+, and HLA-G ^-; CD73 ⁺ , CD105 ⁺ , and CD200 ⁺ ; CD200 ⁺ and OCT-4 ⁺ ; CD73 ^+, CD105 ^+, and HLA-G ^-; CD73 ⁺ and CD105 ⁺ , wherein the population of placental cells is cultured under conditions permitting the formation of embryo-analogs to form one or more embryo-analogs in a population of placental cells comprising said isolated placental cells A group of isolated placental cells that facilitate the stimulation; OCT-4 ⁺ , wherein said population of placental cells is cultured under conditions permitting the formation of embryo-analogs, wherein said isolated placental cells are cultured under conditions that allow for the formation of embryo-analogs, A group of placental cells; Or CD117 ^-, CD133 ^- groups of the isolated placental cells, at least one of and / or ^{PDL1 +, KDR -, CD80 -} , CD86 - -, HLA-A, B, C +, HLA-DP, DQ, DR; Or a combination thereof, wherein at least 70%, 80%, 90%, 95% or 99% of said isolated placental cells are non-maternal origin. In a specific embodiment, the pharmaceutical composition further comprises stem cells not obtained from the placenta.

The compositions provided herein, for example isolated placental cells in a pharmaceutical composition, may comprise placental cells from a single donor or from multiple donors. The isolated placental cells may be fully HLA-matched, or partially or fully HLA-mismatched to the intended recipient.

5.7.4 Matrix containing isolated placental cells

Compositions comprising matrices, hydrogels, scaffolds, etc., comprising a population of placental cells, or isolated placental cells, are further provided herein. Such compositions may be used in place of or in addition to cells in a liquid suspension.

The isolated placental cells described herein can be seeded onto a natural matrix, for example a placental biomaterial, such as a amniotic membrane material. Such amniotic membrane materials include, for example, amniotic membrane dissected directly from the mammalian placenta; Amniotic membrane that is fixed or heat treated, amniotic membrane that is substantially anhydrous (i.e., less than 20% H ₂ O), chorionic membrane, substantially anhydrous chorionic membrane, substantially anhydrous amniotic membrane and chorionic membrane. A preferred placental biomaterial from which isolated placental cells can be seeded is described in U.S. Publication No. 2004/0048796 (Hariri), the disclosure of which is incorporated herein by reference in its entirety.

The isolated placental cells described herein may be suspended, for example, in a hydrogel solution suitable for injection. Suitable hydrogels for such compositions include self-assembling peptides such as RAD16. In one embodiment, the hydrogel solution comprising cells can be cured to form, for example, an injection matrix in which cells are dispersed in the matrix. The isolated placental cells in these matrices can also be cultured so that the cells are expanded by mitosis prior to transplantation. Hydrogels are organic polymers (natural or synthetic) that are crosslinked through covalent bonds, ionic bonds or hydrogen bonds to form gels, for example, to create a three dimensional open lattice structure that captures water molecules. Hydrogel-forming materials include polysaccharides such as alginate and its salts, peptides, polyphosphazines, and polyacrylates (ionically crosslinked), or block polymers such as polyethylene oxide-polypropylene glycol block copolymers (Crosslinked by temperature or pH), respectively. In some embodiments, the hydrogel or matrix is biodegradable.

In some embodiments, the formulation comprises an intramolecular polymeric gel (see, for example, U.S. Patent Application Publication No. 2002/0022676, the disclosure of which is incorporated herein by reference in its entirety); Anseth et al. J. Control Release, 78 (1-3): 199-209 (2002); Wang et al., Biomaterials, 24 (22): 3969-80 (2003)).

In some embodiments, the polymer is at least partially soluble in an aqueous solution, such as water, a buffer salt solution, or an aqueous alcohol solution having a charged side group, or a monovalent salt thereof. Examples of polymers having acidic side groups capable of reacting with cations include poly (phosphazene), poly (acrylic acid), poly (methacrylic acid), copolymers of acrylic acid and methacrylic acid, poly (vinyl acetate) Polymers, such as sulfated polystyrenes. Copolymers having acidic side groups formed by the reaction of acrylic acid or methacrylic acid and vinyl ether monomers or polymers can also be used. Examples of the acidic group are a carboxylic acid group, a sulfonic acid group, a halogenated (preferably fluorinated) alcohol group, a phenolic OH group, and an acidic OH group.

In a specific embodiment, the matrix is a felt which can be made of a bioabsorbable material, for example, PGA, PLA, PCL copolymer or blend, or multifilament yarn made from hyaluronic acid. The multifilament yarns are made of felt using standard fabric processing techniques, including crimping, cutting, carding and needling. In another preferred embodiment, the cells of the invention may be inoculated onto a foam scaffold, which may be a complex structure. In addition, the three-dimensional framework can be molded into a useful shape, for example a specific structure within the body to be restored, replaced or enlarged. Examples of other scaffolds that may be used include nonwoven mat, porous foam, or self-assembled peptides. The nonwoven mat may comprise a fiber (VICRYL, Ethicon, Inc., Somerville, NJ) composed of a synthetic absorbable copolymer of glycolic acid and lactic acid (e.g., PGA / PLA) Can be formed. (See, for example, U.S. Patent No. 6,355,699) formed by a process such as freeze-drying or freeze-drying, for example, composed of a poly (epsilon -caprolactone) / poly (glycolic acid) (PCL / PGA) Can also be used as a scaffold.

The isolated placental cell or co-culture thereof described herein can be seeded onto a three-dimensional framework or scaffold and implanted in vivo. Such frameworks may be implanted in combination with any one or more growth factors, cells, drugs or other ingredients that stimulate tissue formation, for example, to stimulate tissue formation.

Examples of scaffolds that may be used include nonwoven mat, porous foam, or self-assembling peptides. The nonwoven mat may be formed using a fiber (noncryl, eticone, Inc., Somerville, NJ) composed of a synthetic absorbable copolymer of glycolic acid and lactic acid (e.g., PGA / PLA). (See, for example, U.S. Patent No. 6,355,699) formed by a process such as freeze-drying or freeze-drying, for example, composed of a poly (epsilon -caprolactone) / poly (glycolic acid) (PCL / PGA) Can also be used as a scaffold.

In another embodiment, the isolated placental cells are seeded or contacted with a bioabsorbable material such as a PGA, PLA, PCL copolymer or blend, or a felt that may be constructed, for example, by a multifilament yarn made from hyaluronic acid .

In another embodiment, the isolated placental cells provided herein may be seeded onto a foam scaffold, which may be a complex structure. Such a foam scaffold may be shaped into a useful shape, e.g., a shape of a portion of a particular structure within the body to be restored, replaced or enlarged. In some embodiments, the framework is treated with, for example, 0.1 M acetic acid, followed by incubation in polylysine, PBS, and / or collagen, and then cells are inoculated to enhance cell adhesion. The outer surface of the matrix may be coated with a matrix of a plasma-coating or one or more proteins (e.g., collagen, elastic fibers, reticulated fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin- Gelatin, alginate, agar, agarose, and plants, which are not limited to these materials, such as, but not limited to, yeast extract, yeast extract, 4-sulfate, chondroitin-6-sulfate, dermatansulfate, keratin sulfate, By the addition of gum or the like, to improve adhesion or growth of cells and differentiation of tissues.

In some embodiments, the scaffold may comprise or be treated with a material that makes it non-thrombogenic. Such treatments and materials can also promote and maintain endothelial growth, migration, and extracellular matrix deposition. Examples of such materials and treatments include natural materials such as basement membrane proteins such as laminin and type IV collagen, synthetic materials such as EPTFE, and segmented polyurethane silicones such as PURSPAN ™ (The Polymer Technology, Group, Inc., Berkeley, Calif.). The scaffold may also comprise an anti-thrombotic agent such as heparin; The scaffold may also be treated (e. G., Coated with plasma) to alter the surface charge prior to seeding of the isolated placental cells.

The placental cells provided herein (e. G., PDAC) may also be mono-, di-, tri-, beta-tri-, and tetra- calcium phosphate, hydroxyapatite, fluoroapatite, calcium sulfate, calcium fluoride, calcium oxide, calcium carbonate, magnesium calcium phosphates, biologically active glasses such as bio-glass (BIOGLASS) ^®, and mixtures thereof, and inoculated on a ceramic material that is acceptable physiologically are not limited to, in contact with the . Porous biocompatible ceramic materials are present surge (SURGIBONE) ^® (cans Medica Corporation (CanMedica Corp.), Canada), the endo (ENDOBON) ^® (Merck & Bio Materials France (Merck Biomaterial France), France), currently on the market, the three Ross (CEROS) ^® (Mathis, AG (Mathys, AG), Switzerland loom Rahi), and mineralized collagen bone grafting products such as Ross Hill (HEALOS) ™ (for pyuyi, Inc.. (DePuy, Inc.), Massachusetts including the ham lane) and non-Toth (VITOSS) ^®, La course (RHAKOSS) ™, and Cortona's (CORTOSS) ^® (ortho Vita (Orthovita), Malvern, Pennsylvania). The framework may be a mixture, blend or composite of natural and / or synthetic materials.

In one embodiment, the isolated placental cells are seeded or contacted with a suitable scaffold at about 0.5 x 10 ⁶ to about 8 x 10 ⁶ cells / mL.

5.8 Immortal placental cell line

A gene encoding a protein that promotes the growth of transfected cells under suitable conditions so that the production and / or activity of the growth-promoting protein can be regulated by external factors. Lt; RTI ID = 0.0 > PDAC, < / RTI > In a preferred embodiment, the growth-promoting gene is selected from the group consisting of oncogenic genes such as, but not limited to v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1a adenovirus or human papillomavirus E7 protein.

External regulation of the growth-promoting protein may be achieved by altering the composition of the growth-promoting gene to an externally-regulatable promoter, e. G., The temperature of the transfected cell or the medium in contact with the cell, for example under the control of a promoter whose activity can be controlled Can be achieved. In one embodiment, a tetracycline (tet) -controlled gene expression system can be used (Gossen et al., Proc. Natl. Acad. Sci. USA 89: 5547-5551, 1992); [Hoshimaru et al , Proc Natl Acad Sci USA 93: 1518-1523, 1996). In the absence of tet, the tet-controlled transcriptional activator (tTA) in the vector strongly activates transcription from ph _{CMV * -1} (the minimal promoter from human cytomegalovirus fused to the tet operator sequence). The tTA is derived from Escherichia (tetR) of the trans-10-derived tet-resistant operon of E. coli and the fusion protein of the acidic domain of VP16 of the simple herpes virus. the transcriptional activation by tTA is almost completely abolished at a low non-toxic concentration of tet (for example, 0.01 to 1.0 占 퐂 / mL).

In one embodiment, the vector further comprises a gene encoding a selectable marker, e. G., A protein that confers drug resistance. The bacterial neomycin resistance gene (neo ^R ) is one of these markers that can be used in the methods of the invention. cell retaining the neo ^R may be selected by the addition of means known to those skilled in the art, such as, for example 100-200 ㎍ / mL G418 to the growth medium.

Transfection can be accomplished by any of a variety of means known to those skilled in the art, including but not limited to retroviral infection. In general, cell cultures can be transfected by incubation with a mixture of conditioned media and N2 supplemented DMEM / F12 collected from the producer cell line to the vector. For example, placental cell cultures prepared as described above may be incubated in vitro for up to 5 days, for example, by incubation for about 20 hours in DMEM / F12 containing 1 volume of conditioned medium and 2 volumes of N2 supplement It can be infected. The transfected cells carrying the selectable marker can then be selected as described above.

After transfection, the culture is subcultured on a surface that allows proliferation, for example, such that more than 30% of the cells are harvested within a period of 24 hours. Preferably, the substrate is a tissue culture plastic coated with polyornithine (10 / / mL) and / or laminin (10 / / mL), a polylysine / laminin substrate or a polyornithine / laminin It is a substrate. Growth medium, which may or may not have been supplemented with one or more proliferative enhancers, is then fed to the culture every 3 to 4 days. Proliferation enhancers may be added to the growth medium if the culture shows a full culture rate of less than 50%.

When the 80-95% full culture rate has been reached, the placental cell line that has been conditionally immortalized by, for example, trypsin treatment can be subcultured using standard techniques. In some embodiments, it is advantageous to maintain selection until approximately 20 passages (e. G., By the addition of G418 to cells containing the neomycin resistance gene). Cells can also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from conditionally immortalized human placental cell lines prepared as described above. Generally, such clonal cell lines can be isolated and expanded using standard techniques, for example, by limiting dilution or using cloning rings. In general, clonal cell lines can be fed and subcultured as described above.

Conditionally immortalized human placental cell lines, which may be clonal, but need not necessarily be clonal, can be induced to differentiate by inhibiting the production and / or activity of growth-promoting proteins under culturing conditions that generally promote differentiation. For example, when the gene encoding the growth-promoting protein is under the control of an exogenously regulatable promoter, the conditions, for example, the composition or temperature of the medium may be modified to inhibit the transcription of the growth-promoting gene . For the tetracycline-controlled gene expression system discussed above, differentiation can be achieved by adding tetracycline to inhibit the transcription of the growth-promoting gene. Generally, 1 [mu] g / mL tetracycline for 4-5 days is sufficient to initiate differentiation. To facilitate further differentiation, additional agents may be included in the growth medium.

5.9 Kits

In another aspect, tissue-cultured plastic adherent pluripotent placental cells, such as placental stem cells or placental pluripotent cells, such as the cells described in Section 5.2 above (PDAC), and use Kits suitable for the treatment of individuals having a disease or disorder in the circulatory system, including instructions, are provided herein. Preferably, the placental cells are provided in a pharmaceutically acceptable solution, for example a solution suitable for intra-lesional administration or in a solution suitable for intravenous administration. In certain embodiments, placental stem cells or placental pluripotent cells are CD10 ^+, CD34 as described herein ^-, CD105 ⁺ placental cells, e.g., CD10 ^+, CD34 ^-, CD105 ^+, CD200 ⁺ placental cells or CD10 ^+, CD34 ^-, CD45 ^- , CD90 ⁺ , CD105 ⁺ , CD200 ⁺ placental cells.

In certain embodiments, the kit comprises one or more components that facilitate delivery of placental cells to a subject. For example, in certain embodiments, the kit comprises an ingredient that promotes delivery of the placental cells into the lesion to the subject. In such embodiments, the kit may comprise, for example, a syringe and needles suitable for delivery to an individual of the cell. In such embodiments, the placental cells may be contained in a kit in a bag or in one or more vials. In another specific embodiment, the kit comprises an ingredient that facilitates intravenous or intraarterial delivery of placental cells to the subject. In such an embodiment, the placental cells may be contained, for example, in a bottle or bag (e.g., a blood bag or similar bag that may contain up to about 1.5 L of a solution containing cells) Further comprising tubing and needles suitable for delivering the cells.

In addition, the kit may comprise one or more compounds that reduce pain or inflammation in the subject (e.g., analgesic, steroidal or non-steroidal anti-inflammatory compounds, etc.). The kit may comprise an antibacterial or antiviral compound (e.g., one or more antibiotics), a compound that reduces anxiety in the subject (e.g., alapramolam), a compound that reduces immune response in an individual (e.g., A), antihistamines (diphenhydramine, loratadine, desloratadine, quetiapine, fexofenadine, cetirizine, promethazine, chlorpenilamine, levocetirizine, cimetidine, famotidine, ranitidine, nizatidine, ≪ RTI ID = 0.0 > ropacitidine, < / RTI >

In addition, the kit may comprise a single-use article, such as a sterile wipe, disposable paper item, glove, etc., which may facilitate the preparation of the individual for delivery, Thereby reducing the likelihood.

6. Example

6.1 Example 1: phenotypic characterization of placenta-derived adherent cells

This embodiment placental ^cells demonstrates the secretion of angiogenic factors by the (CD10 ^+, CD34, CD105 ^+, CD200 ⁺ placental stem cells, also referred to as the PDAC).

6.1.1 Secretory profiling for evaluation of angiogenic potential of placenta-derived adherent cells

Multiplex bead assay: Placental-derived adherent cells from subculture 6 were plated at the same number of cells in the growth medium, and the conditioned medium was collected after 48 hours. Simultaneous qualitative analysis of multiple angiogenic cytokines / growth factors in cell-conditioned media was performed using a magnetic bead-based multiplex assay (Bio-Flex Pro ™, Bio-Rad, Calif.) , Angiogenesis biomarkers were measured in various matrices including serum, plasma, and cell / tissue culture supernatants. The principle of this 96-well plate-format bead-based assay is similar to the catch sandwich immunoassay. Antibodies designated for target angiogenic targets were covalently coupled to internal stained beads. The coupled beads were reacted with a sample containing an angiogenic target. After removal of unbound proteins by a series of washes, biotinylated detection antibodies specific for the various epitopes were added to the reaction. As a result, a sandwich of antibodies was formed around the angiogenic target. Streptavidin-PE was then added to bind to the biotinylated detection antibody on the bead surface. Briefly, multiplex testing was performed according to the manufacturer's instructions and the amount of each angiogenic growth factor in the conditioned medium was assessed.

ELISA: Quantitation of single angiogenic cytokines / growth factors in cell-conditioned media was performed using a commercially available kit from RAND Systems (Minneapolis, Minn.). Briefly, ELISA assays were performed according to the manufacturer's instructions and the amount of each angiogenic growth factor in the conditioned medium was evaluated.

The level of secretion of various angiogenic proteins by PDAC is shown in Fig.

In a separate experiment, PDAC was also found to secrete angiopoietin-1, angiopoietin-2, PECAM-1 (CD31; platelet endothelial cell adhesion molecule), laminin and fibronectin.

6.2 Example 2: Functional characterization of placental cells

This example demonstrates various characteristics of placental cells (CD10 ⁺ , CD34 ^- , CD105 ⁺ , CD200 ⁺ placental stem cells, also referred to as PDAC) associated with angiogenesis and differentiation potential.

6.2.1 HUVEC tube formation for evaluation of angiogenic potential of PDAC

Human umbilical vein endothelial cells (HUVECs) were subcultured in EGM-2 medium (Cambrex, East Rutherford, NJ) for 3 days to subculture 3 or less and harvested at a full culture rate of approximately 70% -80%. HUVEC was washed once with basal medium / antibiotic (DMEM / F12 (Gibco)) and resuspended to the desired concentration in the same medium. HUVEC was used within 1 hour of manufacture. Human placenta collagen (HPC) was added to a concentration of 1.5 mg / mL in 10 mM HCl (pH 2.25), "neutralized" to pH 7.2 with buffer and kept on ice until use. HPC was combined with the HUVEC suspension at a final cell concentration of 4000 cells / l. The resulting HUVEC / HPC suspension was dispensed immediately into a 96-well plate using a pipette at 3 μl / well (pre-filled with sterile PBS to prevent evaporation around the plate; n = 5 per condition). The HUVEC drops for collagen polymerization without the addition of culture medium and incubated for 75-90 minutes at 37 ℃ and 5% CO _2. At the completion of the "dry" incubation, each well was gently filled with 200 μl of conditioned PDAC medium (n = 2 cell lines) or control medium (eg DMEM / F12 as negative control and EGM-2 as positive control) in, 37 ℃ and 5% CO ₂ and incubated for 20 hours. Prepare conditioned media by incubating PDAC in growth medium for 4-6 hours in growth medium; After attachment and smearing, the medium was changed to DMEM / F12 for 24 hours. After incubation, the medium was removed from the wells without destroying the HUVEC droplets, and the wells were washed once with PBS. The HUVEC droplets were then fixed for 10 seconds, stained with a Diff-Quik cell staining kit for 1 minute, and rinsed 3 times with sterile water. Dyed drops were air dried and images of each well were obtained using a Zeiss SteReo Discovery V8 microscope. The images were then analyzed using a computer software package ImageJ and / or MatLab. Images were converted from color to 8-bit grayscale images and applied to thresholds to convert to monochrome images. The images are then analyzed using the particle analysis feature, which allows for the counting of the values (number of individual particles), total area, average size (of individual particles), and area fraction equal to endothelial tube formation in the assay Pixel density data was obtained.

The conditioned media exerted an angiogenic effect on endothelial cells as evidenced by induction of tubal formation (see Figure 2).

6.2.2 HUVEC migration test

This experiment demonstrated the angiogenic potential of placenta-derived adherent cells. HUVECs were grown to a monolayer culture on fibronectin (FN) -coated 12-well plates and "scratched" with a 1 mL plastic pipette tip onto the monolayer to create a non-foil line across the wells. The "wounded" cells were tested for HUVEC migration by incubation with serum-free conditioned medium (EBM2; Cambrex) from PDAC after 3 days of growth. EBM2 medium containing no cells was used as a control. After 15 hours, cell migration into the acellular region was recorded using an inverted microscope (n = 3). The photographs were then analyzed using computer software packages ImageJ and / or MatLab. Images were converted from color to 8-bit grayscale images and applied to thresholds to convert to monochrome images. The image is then analyzed using the particle analysis feature and the image density (number of individual particles), the total area, the average size (of individual particles), and the area fraction equal to the endothelial transfer in the assay, Data was obtained. The degree of cell migration was scored for the size of the wound line initially recorded, and the results were normalized to 1 x 10 ⁶ cells.

The nutritional factors secreted by placenta-derived adherent cells exerted an angiogenic effect on endothelial cells as evidenced by induction of cell migration (Fig. 3).

In a separate experiment, HUVECs were incubated overnight in the bottom of a 24 well-plate for establishment in EGM2 and then placed in starvation in EBM for half a day. At the same time, the medium-cultured PDAC was thawed and incubated overnight in Transwell (8 [mu] M). After EC starvation, conditioned serum-free DMEM was transferred to the EC with transwell and allowed to proliferate overnight. Each experiment consisted of 4 replicates, and after 24 hours proliferation was assessed by cytometry globulin assay. EBM-2 medium was used as a negative control, and EGM-2 was used as a positive control. The error bars represent the standard deviation of the analytical repeat experiments (n = 3).

The nutritional factors secreted by PDAC caused an increase in the number of HUVEC cells, indicating HUVEC proliferation. Please refer to Fig.

6.2.3 Tubal formation for assessment of angiogenic potential of placenta-derived adherent cells

PDACs were grown in VEGF-free growth medium or VEGF-containing EGM2-MV to assess the effect of VEGF on cell proliferation potential, as well as the angiogenic potential of cells in general. HUVECs were grown in EGM2-MV as control cells for tube formation. Cells were incubated in each medium for 4-7 days until a 70-80% full culture rate was reached. Cold (4 째 C) Matrigel 占 solution (50 쨉 l; BD Biosciences) was dispensed into the wells of a 12-well plate and the plate was incubated for 60 minutes at 37 째 C to gellate the solution. PDAC and HUVEC cells were trypsinized, resuspended in the appropriate medium (containing and without VEGF), and 100 μl of diluted cells (1-3 x 10 ⁴ cells) were added to each of the Matrigel ™ -containing wells Respectively. Cells on polymerized Matrigel ™ in the presence or absence of 0.5-100 ng VEGF were placed in a 5% CO ₂ incubator at 37 ° C for 4-24 hours. After incubation, cells were evaluated for signs of tube formation using standard optical microscopy.

PDAC showed minimal tubule formation in the absence of VEGF, but was induced / differentiated to form tube-like structures through stimulation with VEGF. Please refer to Fig.

6.2.4 Hypoxia Reactivity for Assessing Angiogenic Activity of Adherent Placental Cells

To assess the angiogenic function of endothelial cells and / or endothelial progenitor cells, the cells can be evaluated for their ability to secrete angiogenic growth factors under hypoxic and normal oxygen conditions. Culture under hypoxic conditions generally induces increased secretion of angiogenic factors by endothelial cells or endothelial progenitor cells, which can be measured in conditioned media. Placenta-derived adherent cells were plated at the same cell number in their standard growth medium and grown at approximately 70-80% full culture rate. Subsequently, cells were exchanged with serum-free medium (EBM-2) and incubated for 24 hours under normal oxygen (21% O ₂ ) or hypoxic (1% O ₂ ) conditions. Conditioned media were harvested and the secretion of angiogenic growth factors was analyzed using a commercially available ELISA kit (R & D Systems). ELISA assays were performed according to the manufacturer's instructions and the amount of each angiogenic growth factor (VEGF and IL-8) in the conditioned medium was normalized to 1 x 10 ⁶ cells.

Placenta - derived adherent cells showed elevated secretion of various angiogenic factors under hypoxic conditions. Please refer to Fig.

6.2.5 HUVEC response to PDAC-conditioned media

PDAC for 60 hours in DMEM-LG (Gibco); 40% MCBD-201 (Sigma); 2% FBS (Hyclone Labs), 1x insulin-transferrin-selenium (ITS); 10 ng / mL linoleic acid-bovine serum albumin (LA-BSA); 1 n-dexamethasone (sigma); 100 [mu] M ascorbic acid 2-phosphate (Sigma); 10 ng / mL epidermal growth factor (R & D Systems); And 10 ng / mL platelet-derived growth factor (PDGF-BB) (R & D Systems) and then cultured in serum-free medium for a further 48 hours. Conditioned media from PDAC cultures were collected and used to stimulate serum-starved HUVEC for 5, 15, and 30 minutes. HUVEC was subsequently solubilized and purified using BD ™ CBA Cell Signaling Flex Kit (BD Biosciences) for phosphorylation proteins known to function in angiogenic pathway signaling Lt; / RTI > PDAC has been found to be a strong activator of AKT-1 (inhibits the apoptosis process), AKT-2 (an important signaling protein in the insulin signaling pathway), and ERK1 / 2 cell proliferation pathway in HUVEC. These results further demonstrate the angiogenic potential of PDAC.

6.3 Example 3: Induction of angiogenesis by PDAC

This example demonstrates that PDAC as described in Example 1 above promotes angiogenesis in vivo assays using chick chorioamnion (CAM).

Two separate CAM assays were performed. In the first CAM assay, intact cell pellets from different PDAC preparations were evaluated. In a second CAM assay, the supernatants of different PDAC preparations were evaluated. Fibroblast growth factor (bFGF) was used as a positive control, MDA-MB-231 human breast cancer cells were used as a reference, and vehicle and media controls were used as negative controls. The endpoint of the study was to determine the vascular density of all treated and control groups.

6.3.1 CAM verification using PDAC

PDACs prepared and cryopreserved as described above were used. PDAC was thawed for administration and the number of cells injected onto the CAM was determined.

Study design: The study included 5 groups with 10 embryos in each group. The study design is described in Table 2 below.

CAM assay procedure: Fresh embryos were incubated in a standard egg incubator at 37 占 폚 for 3 days. On day 3, the eggs were broken under sterile conditions and the embryos were placed in 20 100 mm plastic plates and incubated at 37 [deg.] C in an embryo incubator with a water reservoir on the bottom shelf. Air was continually bubbled into the water reservoir using a small pump to keep the humidity in the incubator constant. At a density of: The method of claim 6, a sterile silicon "O" ring, a sterile hood for PDAC 7.69 x 10 ⁵ cells / medium / Matrigel ™ mixtures of 40 ㎕ in the (1: 1), then onto each of the CAM Into each "O" loop. Tables 2A and 2B show the number of cells used and the amount of medium added to each cell sample for administration. 100 ng / ml bFGF in 40 μl DMEM medium / Matrigel ™ mixture (1: 1) for positive control and 40 μl vehicle (PBS / Matrigel ™ 1: 1) for vehicle control embryos 40 [mu] l of DMEM medium was administered. After each administration was terminated, the embryos were returned to the incubator. On day 8, the embryos were removed from the incubator and maintained at room temperature, and the vascular density was determined under each "O" ring using an image capture system at a magnification of 100X.

Vessel density was measured by an angiogenic scoring system using an arithmetic number of 0 to 5 or an exponent number of 1 to 32 to indicate the number of vessels present at the treated site on the CAM. A higher scoring number indicates a higher blood vessel density while a 0 indicates no angiogenesis. Percent inhibition at each administration site was calculated using the score recorded for that site divided by the mean score obtained from the control samples for each individual experiment. The% inhibition for each dose of a given compound was calculated as a collection of all results obtained for that dose from 8-10 embryos.

result

The results of the blood vessel density score are shown in Fig. The results clearly show that the vascular density scores of chick embryo carcinoma treated with PDAC cell suspension, or 100 ng / mL bFGF, or MDAMB231 breast cancer cell suspension, were statistically significantly higher than those of vehicle control CAM (P <0.001 , Student "t" black). The medium used for PDAC culture (negative control) had no effect on vascular density. In addition, the induction of vascular density of PDAC preparations showed some variation, but this variation was not statistically significant.

6.3.2 CAM assay with PDAC supernatant

MDA-MB-231 cells and supernatant samples from PDAC were used for the second CAM assay as described above. The bFGF and MDA-MB-231 supernatants were used as positive controls, and the medium and vehicle were used as negative controls.

Study design: The study included 5 groups with 10 embryos in each group. The study design is described in Table 4 below.

CAM Assay Procedure: The assays were the same as described in Section 6.3.1 above. The only difference is that each stem cell sample or supernatant from MDA-MB-231 cells was used as a test substance. For administration, each supernatant was mixed with Matrigel (1: 1 volume) and 40 [mu] l of the mixture was administered to each embryo.

Results: The vascular density score (see FIG. 8) indicates that the induction of angiogenesis by the supernatant of each stem cell sample was different. The supernatant samples from the PDAC showed significant effects on vessel induction with P < 0.01, P < 0.001 and P < 0.02 (Student "t" assay), respectively. As expected, positive control bFGF also showed strong angiogenesis induction as shown in CAM assay number 1 (P < 0.001, Student &apos; t &apos; However, the supernatant from MDA-MB-231 human breast cancer cells showed no significant induction of angiogenesis relative to the vehicle control. As indicated above, the culture medium alone had no effect.

6.4 Example 4: PDAC shows neuroprotective effect

This example demonstrates that PDAC has a neuroprotective effect at low-oxygen and low-glucose conditions and reduces reactive oxygen species when using an oxygen-glucose deficiency (OGD) damage assay. Thus, these results indicate that PDAC will be useful in treating ischemic conditions, such as stroke or peripheral vascular disease, and will protect against reperfusion injury resulting from ischemic conditions.

Human neurons (ScienCel, catalog # 1520) were cultured according to manufacturer's recommendations. Briefly, the culture vessel was coated with poly-L-lysine (2 [mu] g / mL) in sterile distilled water at 37 [deg.] C for 1 hour. It washed three times containers with double distilled H ₂ O. The neuronal medium (Cyanocell) was added to the vessel and equilibrated in an incubator at 37 占 폚. Neurons were thawed and added directly to the vessel without centrifugation. During the subsequent incubation, the medium was exchanged on the day following initiation of culture, and then exchanged every two days. Neurons are typically prepared for damage by day 4.

OGD medium (Dulbecco's modified Eagle's medium-glucose-free) was prepared by first warming the medium in a water bath to partially reduce the oxygen solubility in the liquid medium. To remove dissolved oxygen, 100% nitrogen was bubbled through the medium for 30 minutes using a 0.5 [mu] m diffuser. HEPES buffer was added to a final concentration of 1 mM. At the end of the aeration, the medium was added directly to the neurons. A small amount of the sample of the medium was sampled to confirm the oxygen level through the dip-type oxygen sensor. The oxygen level is typically reduced from 0.9% to about 5.0% oxygen.

A hypoxic chamber was prepared by placing the chamber in an incubator at 37 [deg.] C for at least 4 hours (preferably overnight) prior to gas treatment. The medium in the culture vessel was removed and replaced with the degassed medium, and the culture vessel was placed in a hypoxic chamber. It was then flushed to a low oxygen chamber with 95% N2 / 5% CO ₂ gas for at least 5 minutes at a speed of 20-25 Lpm through the system. The chamber was degassed one more time after 1 hour, while the system was incubated for 4 hours in an incubator at &lt; RTI ID = 0.0 &gt; 37 C. &lt; / RTI &gt;

At the end of the damage procedure, the OGD medium was aspirated and warmed medium was added to the neurons. After 24-28 hours, PDACs and neurons were plated in equal numbers to 100,000 cells per well of a 6-well plate, neuronal media were added to the neurons and co-cultured for 6 days.

For each condition, a random field in a 6-well plate was photographed. Cells with typical neuronal morphology were identified and neurite length recorded. The average length of neurites correlated positively with neuronal health, and was longer in co-cultures of neurons and PDACs, indicating that PDACs protected cells from damage.

Reactive oxygen species assay

PDAC was determined to express the superoxide dismutase, catalase and heme oxidase genes during hypoxia. The ability of PDAC to remove reactive oxygen species and protect cells from these oxygen species was determined in assays using hydrogen peroxide as a reactive oxygen species inducer.

Description: Target cells (astrocytes; ScienCell Research Laboratories) were inoculated at 6000 / cm ² into 96-well black well plates pre-coated with poly-L-lysine. The astrocytes were stuck overnight in growth medium at 37 ° C with 5% carbon dioxide. The following day, the culture medium was removed, and the cells were incubated with a cell-permeable dye DCFH-DA (dichlorofluorescein diacetate), a fluorescence-generating probe. Surplus dyes were removed by washing with Dulbecco's phosphate buffered saline or Hank's buffered saline solution. The cells were then challenged using reactive oxygen species by the addition of 1000 [mu] M hydrogen peroxide for 30-60 minutes. The hydrogen peroxide-containing medium was then removed and replaced with serum-free, glucose-free growth medium. PDAC was added at 6000 / cm &lt; ² &gt; and the cells were cultured for an additional 24 hours. Cells were then read at 480Ex and 530Em on a standard fluorescent plate reader. The reactive oxygen species content of the medium was directly proportional to the DCFH-DA level in the cytoplasm of the cells. The reactive oxygen species content was measured relative to the pre-determined DCF standard curve. All experiments were performed with N = 24.

For the assay, 1X DCFH-DA was prepared just before use by diluting 1OX of 20X DCFH-DA stock in cell culture medium without fetal bovine serum and stirring to homogeneity. Hydrogen peroxide (H ₂ O ₂ ) dilutions were prepared in DMEM or DPBS if necessary. Standard curves were prepared by diluting 1 mM DCF standards in cell culture medium, transferring 100 [mu] l of DCF standards to a 96 well plate suitable for fluorescence measurement and adding 100 [mu] l cell lysis buffer to a concentration of 0 [mu] M to 10 [ Range as a 1: 10 dilution series. Fluorescence was read at 480Ex and 530Em.

RESULTS: PDAC significantly reduced the concentration of reactive oxygen species in astrocytic co-cultures. See FIG.

6.5 Example 5: Treatment with Placental Stem Cells

6.5.1 Treatment of myocardial infarction

Male subjects in their mid-50s showed chest pain, shortness of breath, nausea, palpitations, and sweating that spread radially to the left arm for more than 20 minutes. Differential diagnosis of myocardial infarction (wall thickening) of the anterior wall of the heart was made using electrocardiographic results and fluctuation of blood concentration of creatine kinase. After stabilizing the object with nitroglycerine and streptomycin kinase object 0.9% brine 1 x 10 ⁸ to about one to 5 x 10 ⁸ of CD10 ^+, CD34 ^-, CD105 ^+, CD200 ⁺ placental stem cells (PDAC) to which the local anesthetic Were administered directly to the affected area using a cardiac syringe. The subjects were then monitored on an emergency basis for 72 hours. The subjects were further monitored over three months after treatment by electrocardiogram and / or dye visualization techniques to assess the degree of vascular remodeling in the infarct region. Therapeutic efficacy is established when electrocardiographic results are close to normal, identifiable as before PDAC administration, or when the infarct zone is visibly re-vascularized at visualization.

6.5.2 Treatment of cardiomyopathy

The subjects showed apnea, leg and ankle swelling, and irregular heartbeats. After excluding the other causes, a diagnosis of cardiomyopathy was made using a confirmatory ECG. Ultrasound images confirmed that the subject had congestive cardiomyopathy. Object of 0.9% saline of 1 x 10 ⁸ to 5 x 10 ⁸ of CD10 ^+, CD34 to a ^- subject to, CD105 ^+, CD200 ⁺ placental stem cells with cardiac syringe with local anesthetic was administered directly into the coronary artery. Individuals were monitored over the next 3 months for changes in ultrasound image readings indicating more normal blood flow, and for improved apnea sensation and reduced leg and ankle swelling. Therapeutic efficacy against an individual is established when any of these indications show improvement during the monitoring period.

6.5.3 Treatment of peripheral vascular disease

The subjects exhibited reddened feet and leg pain, weakness, and fatigue that turned red when hanging. After excluding diabetes, diagnosis of peripheral arterial disease was made. Objects to 1 x 10 mL 0.9% saline of 450 ⁹ to 5 x 10 ⁹ of ^{CD10 +, CD34 -, CD105 +} , CD200 + , and the placental stem cells intravenously, was then monitored every two weeks for three months. Therapeutic efficacy is established when any of the above-described symptoms are improved during the monitoring period.

6.5.4 Treatment of peripheral vascular disease

The subjects exhibited reddened feet and leg pain, weakness, and fatigue that turned red when hanging. After excluding diabetes, diagnosis of peripheral arterial disease was made. I, CD105 ^+, CD200 ⁺ vein placenta stem cells, the administration and / or an equivalent amount of muscle topically between ^toe-object to a 1 x 10 in 5 mL 0.9% saline ⁸ to 5 x 10 ⁸ of CD10 ^+, CD34 Or intraarterially, followed by biweekly monitoring for 3 months thereafter. Therapeutic efficacy is established when any of the above-described symptoms are improved during the monitoring period.

6.5.5 Combination Treatment of Peripheral Vascular Diseases

The subjects exhibited reddened feet and leg pain, weakness, and fatigue that turned red when hanging. After excluding diabetes, diagnosis of peripheral arterial disease was made. Objects to 1 x 10 mL 0.9% saline of 450 ⁹ to 5 x 10 ⁹ of ^{CD10 +, CD34 -, CD105 +} , CD200 + , and the placental stem cells intravenously, was then monitored every two weeks for three months. The subjects were also prescribed to take cilostazol 100 mg twice daily. Therapeutic efficacy is established when any of the above-described symptoms are improved during the monitoring period.

6.5.6 Combination therapy of peripheral vascular disease

The subjects showed painful, weak, and fatigue of the right foot and right leg, which turned red when hanging. After excluding diabetes, diagnosis of peripheral arterial disease was made. The subjects were treated with angioplasty and stent insertion into the femoral artery. Subsequently, to a subject 450 mL 0.9% saline of 1 x 10 ⁹ to 5 x 10 ⁹ of CD10 ^+, CD34 ^- was monitored, CD105 ^+, CD200 ⁺ placental stem cells every two weeks for 3 months after the intravenous administration, and . Therapeutic efficacy is established when any of the above-described symptoms are improved during the monitoring period.

6.5.7 Treatment of a patient with PDAC

A 52-year-old male presented with a left paralysis of the body and partial aphasia. A diagnosis of ischemic stroke was made. After positioning the ischemic region using magnetic resonance imaging, the subject was prepared for surgery to create an opening in the skull on the affected side. After the openings are made, 1-2 mL 0.9% saline solution of 5 x 10 ⁷ to 1 x 10 ⁸ of CD10 ^+, CD34 ^- was administered, CD105 ^+, CD200 ⁺ placental stem cells in the ischemic area. Individuals were monitored for signs of improvement in any symptoms of stroke, especially dysmenorrhoea or aphasia over the next 7-14 days. Therapeutic efficacy is established when any of the above-described symptoms are improved during the monitoring period.

6.5.8 Treatment of a patient with PDAC

A 52-year-old male presented with a left paralysis of the body and partial aphasia. A diagnosis of ischemic stroke was made. After positioning the ischemic region using magnetic resonance imaging, the subject was prepared for surgery to create an opening in the skull on the affected side. After the openings are made, 1 x 10 in 450 mL 0.9% saline solution ⁹ to 5 x 10 ⁹ of CD10 ^+, CD34 ^- a, CD105 ^+, CD200 ⁺ placental stem cells are administered intravenously. Individuals were monitored for signs of improvement in any symptoms of stroke, especially dysmenorrhoea or aphasia over the next 7-14 days. Therapeutic efficacy is established when any of the above-described symptoms are improved during the monitoring period.

Equivalent:

The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the teachings provided herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated herein by reference in their entirety.

Claims (25)

A pharmaceutical composition for treating an object having, CD105 ⁺ and CD200 ⁺ a placental disease or disorder of the circulatory system, comprising a group derived from the adherent cells, ^- determining when CD10 ^+, CD34 by flow cytometry
Wherein the population of cells comprises a sufficient amount for a detectable improvement of one or more symptoms of the disease or disorder in the subject. The pharmaceutical composition according to claim 1, wherein the disease or disorder of the circulatory system is selected from the group consisting of peripheral vascular disease, peripheral arterial disease, diabetic ulcer and severe ischemic ischemia. The pharmaceutical composition according to claim 1, wherein the disease or disorder of the circulatory system is peripheral vascular disease. The pharmaceutical composition according to claim 1, wherein the disease or disorder of the circulatory system is peripheral artery disease. The pharmaceutical composition according to claim 1, wherein the disease or disorder of the circulatory system is a diabetic ulcer. The pharmaceutical composition according to claim 1, wherein the disease or disorder of the circulatory system is severe ischemia. The pharmaceutical composition according to claim 3, wherein the symptom of peripheral vascular disease is reddened palsy, pain, leg weakness and fatigue when hanging. 7. The method according to claim 6, wherein the symptom of severe ischemic injury is selected from the group consisting of pain during ischemic stabilization, severe pain of the legs and feet during immobility of the subject, non-healing wounds on the foot or legs, pain or numbness of the foot, And wherein the composition is flat and dry skin, enlargement of the toenails, absence or reduction of the pulse of the leg or foot, open wound, non-healing skin infection or ulcer, or dry necrosis of the leg or foot. A pharmaceutical composition for treating an object having, CD105 ⁺ and CD200 ⁺ a placental disease or disorder of the circulatory system, comprising a group derived from the adherent cells, ^- determining when CD10 ^+, CD34 by flow cytometry
Wherein said population of cells is contained in an amount sufficient for a detectable improvement in at least one symptom of cardiac function in said subject relative to an individual prior to administration of said placenta-derived adherent cells,
(CO), cardiac index (CI), pulmonary wedge pressure (PAWP),% fractional shortening (% FS), ejection coefficient (EF), left ventricular ejection fraction (LVEF), left ventricular systolic (LVEDD), left ventricular end-diastolic diameter (LVESD), contractility (dP / dt), reduction in atrial or ventricular function, increase in pumping efficiency, decrease in pumping efficiency loss rate, decrease in hemodynamic function loss, A pharmaceutical composition which is reduced in symptoms. As determined by flow cytometry CD10 ^+, CD34 ^-, CD105 ⁺ and CD200 ⁺ the placenta pharmaceutical compositions for therapy or destruction of the object that has the extremities of the peripheral blood flow in the extremities, including the derived adherent cells in a therapeutically effective amount. 11. A pharmaceutical composition according to claim 10 wherein the destruction of blood flow in or around the limb is caused by peripheral arterial disease, severe ischemic or peripheral vascular disease. 11. The pharmaceutical composition according to claim 10, wherein the destruction of blood flow in or around the limb is caused by peripheral vascular disease. 11. The pharmaceutical composition according to claim 10, wherein the destruction of blood flow in or around the limb is caused by peripheral arterial disease. 11. The pharmaceutical composition according to claim 10, wherein the destruction of blood flow in or around the limb is caused by severe ischemia. Any one of claims 1 to 14. A method according to any one of claims, placenta-derived adherent cells are added to the CD45 upon detectable by the flow ^cytometry-in is at least one of CD90 ^+, or a pharmaceutical composition. 15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the placenta-derived adherent cells are further OCT-4 ⁺ upon detection by RT-PCR. 15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the placenta-derived adherent cell composition is administered intravenously, intraarterially, intramuscularly, intradermally or subcutaneously. 16. The pharmaceutical composition according to claim 15, which is administered intravenously, intraarterially, intramuscularly, intradermally or subcutaneously. The pharmaceutical composition according to claim 16, which is administered intravenously, intraarterially, intramuscularly, intradermally or subcutaneously. 15. The method according to any one of claims 1 to 14, further comprising the steps of: 1 x 10 ⁵ , 5 x 10 ⁵ , 1 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ , x 10 ⁸ , 1 x 10 ⁹ , 5 x 10 ^9, or 1 x 10 ¹⁰ placenta-derived adherent cells. 16. The method of claim ^{15, 1 x 10 5, 5 x} 10 5, 1 x 10 6, 5 x 10 6, 1 x 10 7, 5 x 10 7, 1 x 10 8, 5 x 10 8, 1 x 10 9 , 5 x 10 ^9, or 1 x 10 ¹⁰ placenta-derived adherent cells. 17. The method of claim ^{16, 1 x 10 5, 5 x} 10 5, 1 x 10 6, 5 x 10 6, 1 x 10 7, 5 x 10 7, 1 x 10 8, 5 x 10 8, 1 x 10 9 , 5 x 10 ^9, or 1 x 10 ¹⁰ placenta-derived adherent cells. 15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the placenta-derived adherent cells are subcultured at least once prior to administration to the subject. 16. The pharmaceutical composition according to claim 15, wherein the placenta-derived adherent cells have been subcultured at least once before being administered to the subject. 17. The pharmaceutical composition according to claim 16, wherein the placenta-derived adherent cells are subcultured at least once prior to administration to the subject.

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